Compositions for enhanced epithelial permeation of neuropeptide Y for treating obesity

ABSTRACT

Pharmaceutical compositions comprising PYY(3-36) or Neuropeptide Y (NPY), a cyclodextrin, and a compound selected from phosphatidylcholine or diglyceride, wherein the PYY(3-36) or Neuropeptide Y (NPY) is present in an amount effective to alleviate one or more symptom(s) of obesity in a subject, and the cyclodextrin and the compound selected from phosphatidylcholine or diglyceride are present in an amount sufficient to enhance epithelial permeation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation claiming the benefit under 35 U.S.C.§120 of co-pending U.S. patent application Ser. No. 11/561,331, filedNov. 17, 2006; which is a divisional claiming the benefit under 35U.S.C. §120 of U.S. patent application Ser. No. 10/322,266, filed Dec.17, 2002; the contents of this application are hereby incorporated byreference in its entirety.

SEQUENCE LISTING

This application includes a Sequence Listing submitted herewith viaEFS-Web as an ASCII file created on Oct. 22, 2009, named 0204DIV1.txt,which is 201,985 bytes in size, and is hereby incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

A major disadvantage of drug administration by injection is that trainedpersonnel are often required to administer the drug. Forself-administered drugs, many patients are reluctant or unable to givethemselves injections on a regular basis. Injection is also associatedwith increased risks of infection. Other disadvantages of drug injectioninclude variability of delivery results between individuals, as well asunpredictable intensity and duration of drug action.

Despite these noted disadvantages, injection remains the only approveddelivery mode for a large assemblage of important therapeutic compounds.These include conventional drugs, as well as a rapidly expanding list ofpeptide and protein biotherapeutics. Delivery of these compounds viaalternate routes of administration, for example, oral, nasal and othermucosal routes, often yields variable results and adverse side effects,and fails to provide suitable bioavailability. For macromolecularspecies in particular, especially peptide and protein therapeutics,alternate routes of administration are limited by susceptibility toinactivation and poor absorption across mucosal barriers.

Mucosal administration of therapeutic compounds may offer certainadvantages over injection and other modes of administration, for examplein terms of convenience and speed of delivery, as well as by reducing oreliminating compliance problems and side effects that attend delivery byinjection. However, mucosal delivery of biologically active agents islimited by mucosal barrier functions and other factors. For thesereasons, mucosal drug administration typically requires larger amountsof drug than administration by injection. Other therapeutic compounds,including large molecule drugs, peptides and proteins, are oftenrefractory to mucosal delivery.

The ability of drugs to permeate mucosal surfaces, unassisted bydelivery-enhancing agents, appears to be related to a number of factors,including molecular size, lipid solubility, and ionization. Smallmolecules, less than about 300-1,000 daltons, are often capable ofpenetrating mucosal barriers, however, as molecular size increases,permeability decreases rapidly. Lipid-soluble compounds are generallymore permeable through mucosal surfaces than are non-lipid-solublemolecules. Peptides and proteins are poorly lipid soluble, and henceexhibit poor absorption characteristics across mucosal surfaces.

In addition to their poor intrinsic permeability, large macromoleculardrugs, including proteins and peptides, are often subject to limiteddiffusion, as well as lumenal and cellular enzymatic degradation andrapid clearance at mucosal sites. These mucosal sites generally serve asa first line of host defense against pathogens and other adverseenvironmental agents that come into contact with the mucosal surface.Mucosal tissues provide a substantial barrier to the free diffusion ofmacromolecules, while enzymatic activities present in mucosal secretionscan severely limit the bioavailability of therapeutic agents,particularly peptides and proteins. At certain mucosal sites, such asthe nasal mucosa, the typical residence time of proteins and othermacromolecular species delivered is limited, e.g., to about 15-30minutes or less, due to rapid mucociliary clearance.

Various methods and formulations have been attempted to enhance theabsorption of drugs across mucosal surfaces. Penetration enhancingsubstances that facilitate the transport of solutes across biologicalmembranes are widely reported in the art for facilitating mucosal drugdelivery. Mucosal penetration enhancers represented in these reportsinclude (a) chelators (e.g., EDTA, citric acid, salicylates), (b)surfactants (e.g., sodium dodecyl sulfate (SDS)), (c) non-surfactants(e.g., unsaturated cyclic ureas), (d) bile salts (e.g., sodiumdeoxycholate, sodium taurocholate), and (e) fatty acids (e.g., oleicacid, acylcarnitines, mono- and diglycerides). Numerous additionalagents and mechanisms have been proposed for enhancing mucosalpenetration of drugs. These include, for example, reducing the viscosityand/or elasticity of mucus layers that cover mucosal surfaces;facilitating transcellular transport by increasing the fluidity of thelipid bilayer of membranes; altering the physicochemical properties(e.g., lipophilicity, stability) of drugs; facilitating paracellulartransport by altering tight junctions across the epithelial cell layer;overcoming enzymatic barriers; and increasing the thermodynamic activityof candidate drugs.

While many penetration enhancing methods and additives have beenreported to be effective in improving mucosal drug delivery, fewpenetration enhanced products have been developed and approved formucosal delivery of drugs. This failure can be attributed to a varietyof factors, including poor safety profiles relating to mucosalirritation, and undesirable disruption of mucosal barrier functions.

In view of the foregoing, there remains a substantial unmet need in theart for new methods and tools to facilitate mucosal delivery ofbiotherapeutic compounds. Related to this need, there is a compellingneed in the art for methods and formulations to facilitate mucosaldelivery of biotherapeutic compounds that have heretofore provenrefractory to delivery via this route, to avail the medical community ofthe numerous potential advantages of mucosal drug delivery.

One group of therapeutic compounds of interest for mucosal delivery is atherapeutic peptide designated peptide YY. Peptide YY (PYY) as usedherein is a class of peptides, peptide analogs, peptide conjugates andpeptide mimetics exemplified in base structure and activity by aprototypic, 36 amino acid peptide having tyrosine residues at both C-and N-terminals. The paired terminal tyrosine residues in thiswell-known prototypic peptide accounts for the “YY” designation used inthe art. Structural analyses have demonstrated approximately 70%homology between PYY, neuropeptide Y, and pancreatic polypeptide,suggesting a common evolutionary precursor. These three peptidestogether form the primary members of the so-called PancreaticPolypeptide (PP) family believed to play an important role in the normalphysiology of the brain-gut axis. PP peptides all exhibit C-terminalamidation, a feature common in many biologically active peptides.

PYY co-localizes with glucagon and glucagon-like products withinendocrine L cells of the intestinal mucosa and to a lesser extent inalpha cells of the pancreas. Studies in a number of mammals includinghumans have shown that PYY expression increases sequentially along thelength of the intestines, with peptide levels in the rectum up to100-fold greater than in the duodenum. This unique distribution makesPYY an ideal candidate for hormonal regulation of upper gastrointestinalfunction. In fact, PYY causes decreased gastric acid secretion, delaysgastric emptying and slows intestinal transit time. PYY is also known toinhibit exocrine and possibly endocrine functions of the pancreas.

Release of PYY occurs following a meal. An alternate molecular form ofPYY is PYY₃₋₃₆. Eberlein, et al., Peptides 10:797-803, 1989; Eysselein,et al., Peptides 11:111-116, 1990; Grandt, et al., Regul. Pept.51:151-9, 1994, each incorporated herein by reference. This fragmentconstitutes approximately 40% of total PYY-like immunoreactivity inhuman and canine intestinal extracts and about 36% of total plasma PYYimmunoreactivity in a fasting state to slightly over 50% following ameal. It is apparently a dipeptidyl peptidase-IV (DPP4) cleavage productof PYY. PYY₃₋₃₆ is reportedly a selective ligand at the Y2 and Y5receptors, which appear pharmacologically unique in preferringN-terminally truncated (i.e., C-terminal fragments of) NPY analogs.Peripheral administration of PYY reportedly reduces gastric acidsecretion, gastric motility, exocrine pancreatic secretion, gallbladdercontraction and intestinal motility. Yoshinaga, et al., Am. J. Physiol.263:G695-701, 1992; Guan, et al., Endocrinology 128:911-6, 1991; Pappas,et al., Gastroenterology 91:1386-9, 1986; Savage, et al., Gut 28:166-70,1987, each incorporated herein by reference. The effects of centralinjection of PYY on gastric emptying, gastric motility and gastric acidsecretion, as seen after direct injection in or around thehindbrain/brainstem may differ from those effects observed afterperipheral injection. Chen and Rogers, Am. J. Physiol. 269:R787-R792,1995; Chen, et al., Regul. Pept. 61:95-98, 1996; Yang and Tache, Am. J.Physiol. 268:G943-8, 1995; Chen, et al., Neurogastroenterol Motil.9:109-116, 1997, each incorporated herein by reference. For example,centrally administered PYY had some effects opposite to those describedherein for peripherally injected PYY₃₋₃₆ in that gastric acid secretionwas stimulated, not inhibited. Gastric motility was suppressed only inconjunction with TRH stimulation, but not when administered alone, andwas indeed stimulatory at higher doses through presumed interaction withPP receptors. PYY has been shown to stimulate food and water intakeafter central administration. Morley, et al., Brain Res. 341:200-203,1985; Corp, et al., Am. J. Physiol. 259:R317-23, 1990; U.S. ApplicationNo. 20020141985, each incorporated herein by reference.

One of the earliest reported central effects of neuropeptide Y (NPY) wasto increase food intake, particularly in the hypothalamus. Stanley, etal., Peptides 6:1205-11, 1985. PYY and PP are reported to mimic theseeffects, and PYY is more potent or as potent as NPY. Morley, et al.,Brain Res. 341:200-203, 1985; Kanatani, et al., Endocrinology141:1011-6, 2000; Nakajima, et al., J. Pharmacol. Exp. Ther. 268:1010-4,1994, U.S. Application No. 20020141985, each incorporated herein byreference.

Receptors for PYY (designated as Y1, Y2, Y5) have been identifiedthroughout the gastrointestinal tract, including both small bowel andcolon mucosal epithelium. These findings raise the possibility that PYYmay also exhibit additional actions on gastrointestinal tissues,including regulation of cell growth. Dysregulation of cell growth ismost critical in the development and progression of cancer. Prospectiveclinical applications of PYY exist for therapy of malignant disease andcancer. Tseng et al., Peptides 23:389-395, 2002; Michel, et al.,Pharmacol. Rev. 50:143-50, 1998; Gehlert, Proc. Soc. Exp. Biol. Med.218:7-22, 1998, each incorporated herein by reference.

Additional prospective clinical applications of PYY or neuropeptide Yexist for therapy of controlled food intake or obesity. Food intake isregulated by the hypothalamus, including the melanocortin andneuropeptide Y (NPY) systems in the arcuate nucleus. The orexigenic NPYand the anorectic alpha melanocyte-stimulating hormone (α-MSH) systemsof the hypothalamic arcuate nucleus are involved in the centralregulation of appetite. However, the potential mechanisms that linksignaling associated with meal ingestion with these hypothalamic-feedingcircuits are unclear.

From the prototypic PYY peptide, a subpeptide PYY₃₋₃₆ is formed as acleavage product produced by the action of didpetidyl peptidase-IV.PYY₃₋₃₆ is a major circulating species exhibiting a distinctpharmacology, and showing antidiabetic and antiobesity actions inseveral animal models. This gut-derived hormone is releasedpostprandially in proportion to calories ingested. PYY₃₋₃₆ shares 70%amino-acid sequence identity with NPY and acts through NPY receptors.The NPY Y2 receptor (Y2R), a putative inhibitory presynaptic receptor,is highly expressed on NPY neurons in the arcuate nucleus, which isaccessible to peripheral hormones, although not expressed on theneighboring pro-opiomelanocortin (POMC) neurons. Peptide PYY₃₋₃₆, a highaffinity Y2R agonist, is released from the gastrointestinal tractpostprandially in proportion to the calorie content of a meal.

Studies have investigated the effects of peripheral administration ofPYY₃₋₃₆ on feeding. Experiments show that peripheral injection ofPYY₃₋₃₆ in rats inhibits food intake and reduces weight gain. PYY₃₋₃₆also inhibits food intake in mice but not in Y2r-null mice, whichsuggests that the anorectic effect requires the Y2R. Peripheraladministration of PYY₃₋₃₆ increases c-Fos immunoreactivity in thearcuate nucleus and decreases hypothalamic NPY messenger RNA.Intra-arcuate injection of PYY₃₋₃₆ inhibits food intake. PYY₃₋₃₆ alsoinhibits electrical activity of NPY nerve terminals, thus activatingadjacent pro-opiomelanocortin (POMC) neurons. In humans, infusion ofnormal postprandial concentrations of PYY₃₋₃₆ significantly decreasesappetite and reduces food intake by 33% over 24 h. Thus, postprandialelevation of PYY₃₋₃₆ may act through the arcuate nucleus Y2R to inhibitfeeding in a gut-hypothalamic pathway. Nature 418:650-654, 2002,incorporated herein by reference.

Experiments have been performed to investigate whether peripheralPYY₃₋₃₆ might inhibit food intake through the Y2R in the arcuatenucleus, an area that is directly accessible to circulating hormones. Toinvestigate this hypothesis, experiments injected PYY₃₋₃₆ directly intothe arcuate nucleus. In rats fasted for 24 h, food intake wassignificantly decreased by doses as low as 100 fmol, which resulted in asimilar inhibition to that seen after intraperitoneal administration. Toestablish whether these effects occurred through the Y2R, a Y2Rselective agonist, Y2A (N-acetyl [Leu²⁸, Leu³¹] NPY₂₄₋₃₆) [[Y2A(N-acetyl [Leu, Leu] NPY₂₄₋₃₆)]] was used. Its affinity was confirmedusing receptor-binding studies on cell lines that expressed the NPY Y1,Y2 and Y5 receptors. Intra-arcuate nucleus injection of Y2A in ratspreviously fasted for 24 h dose-dependently inhibited (100 fmol to 1nmol) food intake. To confirm the anatomical specificity of this effect,Y2A (100 fmol to 1 nmol) was injected into the paraventricular nucleus(PVN) of rats fasted for 24 h and found no alteration of food intake. Todefine further the role of the Y2R in the feeding inhibition caused byperipheral PYY₃₋₃₆, the effect of PYY₃₋₃₆ on Y2r-null mice andlittermate controls was examined. PYY3-36 inhibited daytime feeding in adose-responsive manner in fasted male wild-type mice but did not inhibitfood intake in fasted male Y2r-null mice. Nature 418:650-654, 2002,incorporated herein by reference.

Results of this experiment suggest that the cells in the arcuate nucleusdetect circulating peripheral satiety signals and relay these signals toother brain regions. This is supported by the observation that leptinmodifies the activity of both the proopiomelanocortin (POMC) and NPYarcuate neurons. Experiments have shown, through a combination ofelectrophysiological and hypothalamic explant studies, that the guthormone, PYY₃₋₃₆, can directly influence hypothalamic circuits, whichresults in coordinate changes in POMC and NPY action. In addition,PYY₃₋₃₆ administered directly into this brain region reduces foodintake. Data show that postprandial concentrations of PYY₃₋₃₆ inhibitfood intake in both rodents and man for up to 12 h, which suggests thatPYY₃₋₃₆ has a role in longer term regulation of food intake. Thiscontrasts with previously characterized gut-derived short-term satietysignals such as cholecystokinin, the effects of which are relativelyshort-lived. The failure of PYY₃₋₃₆ to inhibit food intake in theY2r-null mice provides further evidence that PYY₃₋₃₆ reduces food intakethrough a Y2R-dependent mechanism. Experimental results suggest that agut-hypothalamic pathway that involves postprandial PYY₃₋₃₆ acting atthe arcuate Y2R has a role in regulating feeding. Thus, the PYY₃₋₃₆system may provide a therapeutic target for the treatment of obesity.Nature 418:650-654, 2002, incorporated herein by reference.

Leptin is an adiposity hormone that modulates the activity of multiplehypothalamic signaling pathways involved in the control of food intake.Experiments were designed to evaluate whether central administration ofleptin or one of its downstream mediators, neuropeptide Y (NPY), couldaffect food intake by modulating the brain stem neurophysiologicalresponse to ascending meal-related feedback signals in the nucleus ofthe solitary tract (NTS) in anesthetized male Long-Evans rats. NTSneurons at the rostrocaudal level of the area postrema weredose-dependently activated by gastric loads ranging from 2-10 ml, andleptin and NPY had opposite modulatory effects on this loadvolume/activity relationship: leptin significantly increased NTSresponses to gastric loads, whereas NPY reduced the potency and efficacywith which gastric loads activated NTS neurons. These effects wereprobably not mediated by peripheral effects of centrally administeredpeptides or by the gastrokinetic effects of central NPY or leptin,because the dose-response relationship between gastric load volume andneurophysiological firing rate was unchanged in gastric load-sensitivevagal afferent fibers. These data suggest a mechanistic framework forconsidering how feeding behavior occurring in meals is altered bychallenges to energy homeostasis, such as fasting and overfeeding.Endocrinology 143:3779-3784, 2002, incorporated herein by reference.

As noted above, the subpeptide PYY₃₋₃₆ exhibits antidiabetic andantiobesity actions in several animal models. Since gastric emptying isan important mediator of post-prandial glycemia, experimentsinvestigated whether PYY₃₋₃₆ affected gastric emptying, and specificallyinvestigated whether such effect was mediated via the area postrema(AP), a circumventricular organ with no blood-brain barrier, that isaccessible to circulating peptides and known to be involved in theregulation of gastrointestinal function. While saline injectedAP-lesioned animals had a tendency to delay gastric emptying compared tonon-operated and sham operated rats, PYY₃₋₃₆ administration had noadditional effect on gastric emptying rate in the AP-lesioned animals.PYY₃₋₃₆ has a potent effect to inhibit gastric emptying in normal ratsvia a pathway that appears to include the area postrema. AmericanDiabetes Association, 62^(nd) Annual Scientific Sessions, Jun. 14-18,2002; Abstract 1661-P, incorporated herein by reference.

To investigate a possible role for peripherally administered PYY₃₋₃₆ inmetabolic control, experiments examined the effects of its infusion for28 days via subcutaneous osmotic pumps (at 0, 30, 100, 300 and 1000μg/kg/d) to C57BL/6 mice (n=14-22/group) previously fed a high fat diet(HF; 58% fat vs LF, low-fat control diet, with 11% fat) for 47 days.Contrasting with previously reported effects of centrally administeredPYY and PYY₃₋₃₆ to increase food intake and body weight, the results ofthis study indicate that peripherally administered PYY₃₋₃₆ exhibits ananti-obesity and glucose-lowering effect in diet-induced obese mice.American Diabetes Association, 62^(nd) Annual Scientific Sessions, Jun.14-18, 2002; Abstract 1718-P, incorporated herein by reference.

Additional studies examined the effect of 4 weeks of continuous infusionof PYY₃₋₃₆ on 8 week-old male Zucker Diabetic Fatty (ZDF) rats. Theresults suggest that the improvement in glycemic control followingchronic PYY₃₋₃₆ in ZDF rats was unlikely to be due to direct effects onglucoregulatory hormone secretion, and may instead be related tomechanisms such as reduced nutrient availability. The dissociation offood consumption from weight gain in these experiments, despite thepresence of glucose-lowering effects, suggests factors such as caloricloss in the urine of diabetic animals. Further studies in Diabetic FattyZucker (ZDF) rats demonstrated that continuous infusion of PYY₃₋₃₆decreased food consumption and improved glycemic control, withoutdecreasing body weight gain, but those observations did not account forcaloric redistribution due to treatment-related changes in glycosuria.In contrast to previous data in diabetic animals where caloric losses inthe urine may have been a factor and where there was no change in bodyweight, the results of the present study indicate that in the absence ofglycosuria in obese non-diabetic fa/fa rats, PYY₃₋₃₆ treatment decreasesfood consumption and body weight. American Diabetes Association, 62^(nd)Annual Scientific Sessions, Jun. 14-18, 2002; Abstract 1717-P; Abstract2499, incorporated herein by reference.

Further experiments examined the effect of continuous infusion ofPYY₃₋₃₆ in genetically obese mice (ob/ob; B6.V-Lep ob) on food intakeand body. Chronic PYY₃₋₃₆ infusion in ob/ob mice was associated with adose dependent decrease in body weight compared to saline infusedanimals. This effect appeared to be independent of any effect on foodintake. American Diabetes Association, 62^(nd) Annual ScientificSessions, Jun. 14-18, 2002; Abstract 1716-P, incorporated herein byreference.

As part of an investigation of the potential involvement of the gut inthese actions, other experiments examined the dose-response ofsubcutaneously injected PYY₃₋₃₆ on pentagastrin-stimulated gastric acidsecretion. The effect of PYY₃₋₃₆ on ethanol-induced gastritis wasevaluated in fasted male Sprague Dawley rats. To study acid secretion,rats chronically implanted with gastric cannulae were injectedsubcutaneously with 125 μg/kg pentagastrin. Gastric contents obtained byflushing the cannulae every 10 min were titrated to measure acidproduction. PYY₃₋₃₆ dose-dependently and potently inhibitedpentagastrin-stimulated gastric acid secretion, manifest also as agastroprotective effect, in rats. Endogenously circulating PYY₃₋₃₆ mayplay a physiologic role in controlling gastric acid secretion andprotecting the gastric mucosa. American Diabetes Association, 62^(nd)Annual Scientific Sessions, Jun. 14-18, 2002; Abstract 2444-PO,incorporated herein by reference.

Obesity and its associated disorders are common and very serious publichealth problems in the United States and throughout the world. Upperbody obesity is the strongest risk factor known for type 2 diabetesmellitus, and is a strong risk factor for cardiovascular disease.Obesity is a recognized risk factor for hypertension, arteriosclerosis,congestive heart failure, stroke, gallbladder disease, osteoarthritis,sleep apnea, reproductive disorders such as polycystic ovarian syndrome,cancers of the breast, prostate, and colon, and increased incidence ofcomplications of general anesthesia. Kopelman, Nature 404:635-43, 2000,incorporated herein by reference. It reduces life-span and carries aserious risk of co-morbidities above, as well disorders such asinfections, varicose veins, acanthosis nigricans, eczema, exerciseintolerance, insulin resistance, hypertension hypercholesterolemia,cholelithiasis, orthopedic injury, and thromboembolic disease. Rissanen,et al., BMJ 301:835-7, 1990, incorporated herein by reference. Obesityis also a risk factor for the group of conditions called insulinresistance syndrome, or “Syndrome X.” Recent estimates for the medicalcost of obesity and associated disorders are $150 billion worldwide. Thepathogenesis of obesity is believed to be multifactorial but the basicproblem is that in obese subjects nutrient availability and energyexpenditure do not come into balance until there is excess adiposetissue. Obesity is currently a poorly treatable, chronic, essentiallyintractable metabolic disorder. A therapeutic drug useful in weightreduction of obese persons could have a profound beneficial effect ontheir health.

Previous methods have attempted to increase weight gain in healthysubjects by administering peptide YY to the subject. Additional reportspropose treatment of metabolic disorders such as obesity, diabetes, andincreased cardiovascular risk by administering peptide YY or a peptideYY agonist. See e.g., U.S. Pat. No. 5,912,227; U.S. Application No.20020141985. These and related reports have attempted to administerpeptide YY by various routes. Notably, there are no previousdescriptions that demonstrate a therapeutically effective nasalformulation or method for administering peptide YY for any of theforegoing diseases and conditions.

Previous attempts to successfully deliver peptide YY for therapeuticpurposes have suffered from a number of important and confoundingdeficiencies. These deficiencies point to a long-standing unmet need inthe art for pharmaceutical formulations and methods of administeringpeptide YY that are stable and well tolerated and that provide enhanceddelivery to target sites, e.g., the serum or other selected cellular orextracellular compartment, in mammalian subjects.

BRIEF SUMMARY OF THE INVENTION

The present invention fulfills the foregoing needs and satisfiesadditional objects and advantages by providing novel, effective methodsand compositions for intranasal delivery of peptide YY yielding improvedpharmacokinetic and pharmacodynamic results. In certain aspects of theinvention, the peptide YY is delivered to the intranasal mucosa alongwith one or more intranasal delivery-enhancing agent(s) to yieldsubstantially increased absorption and/or bioavailability of the peptideYY and/or a substantially decreased time to maximal concentration ofpeptide YY in a tissue or other target site of a mammalian subject ascompared to controls (e.g., where the peptide YY is administered to thesame intranasal site alone or formulated according to previouslydisclosed reports).

The enhancement of intranasal delivery of peptide YY according to themethods and compositions of the invention allows for the effectivepharmaceutical use of these agents to treat a variety of diseases andconditions in mammalian subjects.

Briefly, the methods and compositions of the invention provide forenhanced delivery of peptide YY across nasal mucosal barriers to reachnovel target sites for drug action yielding an enhanced, therapeuticallyeffective rate or concentration of delivery. In certain aspects,employment of one or more intranasal delivery-enhancing agentsfacilitates the effective delivery of a peptide YY to a targeted,extracellular or cellular compartment, for example the systemiccirculation, a selected cell population, tissue or organ. Exemplarytargets for enhanced delivery in this context are target physiologicalcompartments, tissues, organs and fluids (e.g., within the blood serum,central nervous system (CNS) or cerebral spinal fluid (CSF) or selectedtissues or cells of the liver, bone, muscle, cartilage, pituitary,hypothalamus, kidney, lung, heart, testes, skin, or peripheral nervoussystem.

In exemplary embodiments, the enhanced delivery methods and compositionsof the present invention provide for therapeutically effective mucosaldelivery of peptide YY for prevention or treatment of obesity and eatingdisorders in mammalian subjects. In one aspect of the invention,pharmaceutical formulations suitable for intranasal administration areprovided that comprise a therapeutically effective amount of peptide YYand one or more intranasal delivery-enhancing agents as describedherein, which formulations are effective in a nasal mucosal deliverymethod of the invention to prevent the onset or progression of obesityor eating disorders in a mammalian subject. Nasal mucosal delivery of atherapeutically effective amount of peptide YY and one or moreintranasal delivery-enhancing agents yields more consistent (normalized)or elevated therapeutic levels of peptide YY in a central nervous systemtissue or fluid. In an exemplary embodiment, nasal mucosal delivery of atherapeutically effective amount of peptide YY and one or moreintranasal delivery-enhancing agents yields more consistent (normalized)or elevated therapeutic levels of peptide YY in a region of the brain ofthe mammalian subject, for example, hypothalamus, or arcuate nucleus ofthe hypothalamus. Nasal mucosal delivery of a therapeutically effectiveamount of peptide YY and one or more intranasal delivery-enhancingagents yields elevated therapeutic levels of peptide YY in the subjectand inhibits food intake in the mammalian subject, reducing symptoms ofobesity or an eating disorder.

In other embodiments, the enhanced delivery methods and compositions ofthe present invention provide for therapeutically effective mucosaldelivery of a neuropeptide Y receptor antagonist, for example, a Y1receptor antagonist, for prevention or treatment of obesity and eatingdisorders in mammalian subjects. In one aspect of the invention,pharmaceutical formulations suitable for intranasal administration areprovided that comprise a therapeutically effective amount of the Y1receptor antagonist, for example, a dihydropyridine or peptide YYanalog, and one or more intranasal delivery-enhancing agents asdescribed herein, which formulations are effective in a nasal mucosaldelivery method of the invention to prevent the onset or progression ofobesity or eating disorders in a mammalian subject. Nasal mucosaldelivery of a therapeutically effective amount of Y1 receptor antagonistand one or more intranasal delivery-enhancing agents yields moreconsistent, e.g., normalized, or elevated therapeutic levels of peptideYY in a central nervous system tissue or fluid or in a blood serum ofthe mammalian subject.

The enhanced delivery methods and compositions of the present inventionprovide for therapeutically effective mucosal delivery of peptide YY forprevention or treatment of a variety of diseases and conditions inmammalian subjects. Peptide YY can be administered via a variety ofmucosal routes, for example by contacting peptide YY to a nasal mucosalepithelium, a bronchial or pulmonary mucosal epithelium, an oral,gastric, intestinal or rectal mucosal epithelium, or a vaginal mucosalepithelium. In exemplary embodiments, the methods and compositions aredirected to or formulated for intranasal delivery (e.g., nasal mucosaldelivery or intranasal mucosal delivery).

In one aspect of the invention, pharmaceutical formulations suitable forintranasal administration are provided that comprise a therapeuticallyeffective amount of peptide YY and one or more intranasaldelivery-enhancing agents as described herein, which formulations areeffective in a nasal mucosal delivery method of the invention to preventthe onset or progression of obesity, cancer, or malnutrition or wastingrelated to cancer in a mammalian subject, or to alleviate one or moreclinically well-recognized symptoms of obesity, cancer, or malnutritionor wasting related to cancer in a mammalian subject.

In another aspect of the invention, pharmaceutical formulations suitablefor intranasal administration are provided that comprise atherapeutically effective amount of peptide YY and one or moreintranasal delivery-enhancing agents as described herein, whichformulation is effective in a nasal mucosal delivery method of theinvention to alleviate symptoms or prevent the onset or lower theincidence or severity of, for example, obesity in children or adultsubjects, Alzheimer's disease, colon carcinoma, colon adenocarcinoma,pancreatic carcinoma, pancreatic adenocarcinoma, breast carcinoma,treatment and prevention of malnutrition resulting from iatrogeniccauses or cachexia associated with advanced disease, or cancer cachexia.

In another aspect of the invention, pharmaceutical formulations andmethods are directed to administration of peptide YY in combination withvitamin E succinate. Peptide YY in combination with vitamin E succinatemay be administered to alleviate symptoms or prevent the onset or lowerthe incidence or severity of cancer, for example, colon adenocarcinoma,pancreatic adenocarcinoma, or breast cancer.

In more detailed aspects of the invention, methods and compositions forintranasal delivery of peptide YY incorporate one or more intranasaldelivery enhancing agent(s) combined in a pharmaceutical formulationtogether with, or administered in a coordinate nasal mucosal deliveryprotocol with, a therapeutically effective amount of peptide YY. Thesemethods and compositions provide enhanced nasal transmucosal delivery ofthe peptide YY, often in a pulsatile delivery mode to maintain continuedrelease of peptide YY to yield more consistent (normalized) or elevatedtherapeutic levels of peptide YY in the blood serum, or in anotherselected physiological compartment or target tissue or organ fortreatment of disease. Peptide YY is produced in the gastrointestinaltract and brain. A major site of action of peptide YY in the brain isthe hypothalamic arcuate nucleus. For example, normalized and elevatedtherapeutic levels of peptide YY may be measured in the central nervoussystem tissue or cerebral spinal fluid or in the systemic blood serum ofthe mammalian subject receiving the peptide YY by enhanced nasaltransmucosal delivery using methods and compositions of the presentinvention. Normalized and elevated therapeutic levels of peptide YY aredetermined, for example, by an increase in bioavailability (e.g., asmeasured by maximal concentration (C_(max)) or the area underconcentration vs. time curve (AUC) for an intranasal effective amount ofpeptide YY) and/or an increase in delivery rate (e.g., as measured bytime to maximal concentration (t_(max)), C_(max), and or AUC).Normalized and elevated high therapeutic levels of peptide YY in thecentral nervous system (CNS) tissue or fluid or in the blood serum maybe achieved in part by repeated intranasal administration to a subjectwithin a selected dosage period, for example an 8, 12, or 24 hour dosageperiod.

In an alternative embodiment, normalized and elevated therapeutic levelsof peptide YY are determined, for example, by an increase inbioavailability and/or an increase in delivery rate as measured in thehepatic portal vein, (e.g., as measured by t_(max), C_(max), or AUC foran intranasal effective amount of peptide YY in the hepatic portalvein).

To maintain more consistent or normalized therapeutic levels of peptideYY, the pharmaceutical formulations of the present invention are oftenrepeatedly administered to the nasal mucosa of the subject, for example,one, two or more times within a 24 hour period, four or more timeswithin a 24 hour period, six or more times within a 24 hour period, oreight or more times within a 24 hour period. The methods andcompositions of the present invention yield improved pulsatile deliveryto maintain normalized and/or elevated therapeutic levels of peptide YY,e.g., in the blood serum. The methods and compositions of the inventionenhance transnasal mucosal delivery of peptide YY to a selected targettissue or compartment by at least a two- to five-fold increase, moretypically a five- to ten-fold increase, and commonly a ten- totwenty-five- up to a fifty-fold increase (e.g., as measured by t_(max)C_(max), and/or AUC, in the central nervous system (CNS) tissue or fluidor the blood serum, or in another selected physiological compartment ortarget tissue or organ for delivery), compared to the efficacy ofdelivery of peptide YY administered alone or using apreviously-described delivery method, for example a previously-describedmucosal delivery, intramuscular delivery, subcutaneous delivery,intravenous delivery, and/or parenteral delivery method.

Nasal mucosal delivery of peptide YY according to the methods andcompositions of the invention will often yield effective delivery andbioavailability that approximates dosing achieved by continuousadministration methods. In other aspects, the invention providesenhanced nasal mucosal delivery that permits the use of a lower systemicdosage and significantly reduces the incidence of peptide YY-relatedside effects. Because continuous infusion of peptide YY outside thehospital setting is otherwise impractical, mucosal delivery of peptideYY as provided herein yields unexpected advantages that allow sustaineddelivery of peptide YY, with the accrued benefits, for example, ofimproved patient-to-patient dose variability.

In more detailed aspects of the invention, the methods and compositionsof the present invention provide improved and/or sustained delivery ofpeptide YY to the blood serum or central nervous system (CNS). In oneexemplary embodiment, an intranasal effective amount of peptide YY andone or more intranasal delivery enhancing agent(s) is contacted with anasal mucosal surface of a subject to yield enhanced mucosal delivery ofpeptide YY to the blood serum or CNS of the subject, for example, toeffectively treat obesity, cancer, or malnutrition or wasting related tocancer. In certain embodiments, the methods and compositions of theinvention provide improved and sustained delivery of peptide YY to bloodserum or CNS of peptide YY action, including the central nervous system(CNS) or cerebral spinal fluid (CSF) of the subject, and willeffectively treat one or more symptoms of obesity, cancer, ormalnutrition or wasting related to cancer, including in cases whereconventional peptide YY therapy yields poor results or unacceptableadverse side effects.

In exemplary embodiments, the methods and compositions of the presentinvention yield a two- to five-fold decrease, more typically a five- toten-fold decrease, and commonly a ten- to twenty-five- up to a fifty- toone hundred-fold decrease in the time to maximal concentration (t_(max))of the peptide YY in blood serum, gastrointestinal tract, centralnervous system, cerebral spinal fluid, and/or in another selectedphysiological compartment or target tissue or organ for delivery—ascompared to delivery rates for peptide YY administered alone or inaccordance with previously-described drug delivery methods. Suchpreviously described methods may include, for example, any alternateroute of deliver (e.g., intravenous or subcutaneous versus intranasal).

In further exemplary embodiments, the methods and compositions of theinvention yield a two- to five-fold increase, more typically a five- toten-fold increase, and commonly a ten- to twenty-five- up to a fifty- toone hundred-fold increase in the area under concentration vs. timecurve, AUC, of the peptide YY in blood serum, gastrointestinal tract,central nervous system, cerebral spinal fluid, or in another selectedphysiological compartment or target tissue or organ for delivery—ascompared to delivery rates for the peptide YY administered alone or inaccordance with previously-described administration methods.

In further exemplary embodiments, the methods and compositions of thepresent invention yield a two- to five-fold increase, more typically afive- to ten-fold increase, and commonly a ten- to twenty-five- up to afifty- to one hundred-fold increase in the maximal concentration,C_(max), of the peptide YY in blood serum, CNS, gastrointestinal tract,or in another selected physiological compartment or target tissue ororgan for delivery—as compared to delivery rates for the peptide YYadministered alone or in accordance with previously-describedadministration methods.

The methods and compositions of the invention will often serve toimprove peptide YY dosing schedules and thereby maintain normalizedand/or elevated, therapeutic levels of peptide YY in the subject. Incertain embodiments, the invention provides compositions and methods forintranasal delivery of peptide YY, wherein peptide YY dosage normalizedand sustained by repeated, typically pulsatile, delivery to maintainmore consistent, and in some cases elevated, therapeutic levels. Inexemplary embodiments, the time to maximum concentration (t_(max)) ofpeptide YY in the blood serum, CNS, or gastrointestinal tract will befrom about 0.1 to 4.0 hours, alternatively from about 0.4 to 1.5 hours,and in other embodiments from about 0.7 to 1.5 hours, or from about 1.0to 1.3 hours. Thus, repeated intranasal dosing with the formulations ofthe invention, on a schedule ranging from about 0.1 to 2.0 hours betweendoses, will maintain normalized, sustained therapeutic levels of peptideYY to maximize clinical benefits while minimizing the risks of excessiveexposure and side effects.

The methods and compositions of the invention will often serve toimprove peptide YY dosing schedules and thereby maintain normalizedand/or elevated, therapeutic levels of peptide YY in the subject. Inexemplary embodiments, dosage of peptide YY is from about 0.001 pmol toabout 100 pmol per kg body weight, from about 0.01 pmol to about 10 pmolper kg body weight, from about 0.1 pmol to about 5 pmol per kg bodyweight. In further exemplary embodiments, dosage of peptide YY is fromabout 0.5 pmol to about 1.0 pmol per kg body weight.

In an alternative embodiment, the invention provides compositions andmethods for intranasal delivery of peptide YY, wherein the peptide YYcompound(s) is/are repeatedly administered through an intranasaleffective dosage regimen that involves multiple administrations of thepeptide YY compound(s) to the subject during a daily or weekly scheduleto maintain a therapeutically effective elevated and lowered pulsatilelevel of peptide YY during an extended dosing period. The compositionsand method provide peptide YY compound(s) that are self-administered bythe subject in a nasal formulation between two and six times daily tomaintain a therapeutically effective elevated and lowered pulsatilelevel of peptide YY during an 8 hour to 24 hour extended dosing period.

In alternative embodiments, the invention achieves enhanced delivery ofnormalized and/or elevated, improved therapeutic levels of peptide YY bycombining mucosal administration of one dosage amount of peptide YYformulated with one or more intranasal delivery-enhancing agents, with aseparate dosage amount of peptide YY delivered by a non-mucosal route,for example by subcutaneous or intramuscular administration. In oneexemplary embodiment, intranasal delivery of peptide YY according to thecompositions and methods herein yields normalized and/or elevated, hightherapeutic levels of peptide YY in the blood serum, CNS, orgastrointestinal tract of the subject for a time period betweenapproximately 0.1 and 3 hours following intranasal administration.Coordinate administration of peptide YY by a non-mucosal route (before,simultaneous with, or after mucosal administration) provides moreconsistent, elevated therapeutic levels of peptide YY in the bloodserum, CNS, or gastrointestinal tract of the subject for an effectivetime period of between approximately 2 to 24 hours, more often betweenabout 4 to 16 hours, and in certain embodiments between about 6 to 8hours. Within these coordinate administration methods, the aims of thetreating physician are facilitated by improving clinical benefit whileminimizing the risks of excessive exposure.

In other aspects of the invention, the methods and formulations forintranasally administering peptide YY described herein yield asignificantly enhanced rate or level of delivery (e.g., decreasedt_(max), increased AUC, and/or increased C_(max)) of the peptide YY intothe serum, or to selected tissues or cells, of the subject. Thisincludes enhanced delivery rates or levels into the blood serum, CNStissue or fluid, or to selected tissues or cells (e.g., blood serum,CNS, or CSF), compared to delivery rates and levels for the peptide YYadministered alone or in accordance with conventional technologies.Thus, in certain aspects of the invention, the foregoing methods andcompositions are administered to a mammalian subject to yield enhanceddelivery of the peptide YY to a physiological compartment, fluid, tissueor cell within the mammalian subject.

Within more detailed aspects of the invention, bioavailability ofpeptide YY achieved by the methods and formulations herein (e.g.,measured by peak blood plasma levels (C_(max)) in blood serum or inanother selected physiological compartment or target tissue) will be,for example, from about 0.1 pmol/L to about 1000 pmol/L of blood plasmaor CSF, from about 1.0 pmol/L to about 100 pmol/L of blood plasma orCSF, from about 1.0 pmol/L to about 10 pmol/L of blood plasma or CSF, orfrom about 5.0 pmol/L to about 10 pmol/L of blood plasma or CSF.

Within other detailed aspects of the invention, bioavailability ofpeptide YY achieved by the methods and formulations herein (e.g., asmeasured by area under the concentration curve (AUC) in blood plasma,central nervous system (CNS) tissue or fluid, or in another selectedphysiological compartment or target tissue) will be, for example, fromabout 0.1 pmol/L to about 1000 pmol/L of blood plasma or CSF, from about10 pmol/L to about 1000 pmol/L of blood plasma or CSF, from about 10pmol/L to about 100 pmol/L of blood plasma or CSF, or from about 50pmol/L to about 100 pmol/L of blood plasma or CSF

The procedures for determining the concentrations of peptide YYneuropeptide Y, and pancreatic peptide in blood serum, central nervoussystem (CNS) tissues or fluids, cerebral spinal fluid (CSF), or othertissues or fluids of a mammalian subject may be determined byimmunologic assay for peptide YY neuropeptide Y, and pancreatic peptide.The procedures for determining the concentrations of peptide YYneuropeptide Y, and pancreatic peptide as test materials for evaluatingenhanced permeation of active agents in conjunction with coordinateadministration of mucosal delivery-enhancing agents or combinatorialformulation of the invention are generally as described above and inaccordance with known methods and specific manufacturer instructions forradioimmunoassay (RIA), enzyme immunoassay (EIA), and antibody reagentsfor immunohistochemistry or immunofluorescence for peptide YYneuropeptide Y, or pancreatic peptide. Bachem, AG (King of Prussia,Pa.).

The procedures for determining the concentrations of neuropeptide Yreceptors Y1 through Y5 as test materials for evaluating enhancedpermeation of active agents in conjunction with coordinateadministration of mucosal delivery-enhancing agents or combinatorialformulation of the invention are generally as described above and inaccordance with known methods and specific manufacturer instructions forassays to measure neuropeptide Y receptor binding by a neuropeptide YScintillation Proximity (SPA) Binding Assay [Receptors Y1; Y2]; AmershamBiosciences (Piscataway, N.J.).

Within further detailed aspects of the invention, bioavailability ofpeptide YY achieved by the methods and formulations herein (e.g., asmeasured by time to maximal concentration (t_(max)) in central nervoussystem (CNS) tissue or fluid, blood serum, or in another selectedphysiological compartment or target tissue) will be, for example, about1.4 hours or less, about 1.0 hours or less, about 0.8 hours or less,about 0.6 hours or less, about 0.4 hours or less, or about 0.2 hours orless.

In exemplary embodiments, administration of one or more peptide YYformulated with one or more intranasal delivery-enhancing agents asdescribed herein yields effective delivery to the central nervous system(CNS) tissue or fluid, or to the blood serum to alleviate a selecteddisease or condition (e.g., obesity, cancer, or malnutrition or wastingrelated to cancer, or a symptom thereof) in a mammalian subject. In moredetailed aspects, the methods and formulations for intranasallyadministering peptide YY according to the invention yield asignificantly enhanced rate or level of delivery (e.g., decreasedt_(max) or increased C_(max)) of the peptide YY into the serum or toselected tissues or cells (e.g., gastrointestinal tract or CNS),compared to delivery rates and levels for the peptide YY administeredalone or in accordance with previously-described technologies.

The enhanced pharmacokinetics of delivery of peptide YY (e.g., increasedrate, normalized, sustained delivery, and elevated levels) according tothe methods of the invention, provides improved therapeutic efficacy,e.g., to treat obesity, cancer, or malnutrition or wasting related tocancer in a subject, without unacceptable adverse side effects. Thus,for example, pharmaceutical preparations formulated for nasal mucosaldelivery are provided for treating obesity, cancer, or malnutrition orwasting related to cancer in a mammalian subject that comprise atherapeutic intranasal effective amount of peptide YY combined with oneor more intranasal delivery-enhancing agents as disclosed herein. Thesepreparations surprisingly yield enhanced mucosal absorption of thepeptide YY to produce a therapeutic effective concentration of the drug(e.g., for treating obesity, cancer, or malnutrition or wasting relatedto cancer in a subject) at a target site or tissue in the subject inabout 45 minutes or less, 30 minutes or less, 20 minutes or less, or aslittle as 15 minutes or less.

Within other detailed embodiments of the invention, the foregoingmethods and formulations are administered to a mammalian subject toyield enhanced blood serum, gastrointestinal tract, CNS levels, or othertissue levels of the peptide YY by administering a formulationcomprising an intranasal effective amount of peptide YY and one or moreintranasal delivery-enhancing agents and one or more sustainedrelease-enhancing agents. The sustained release-enhancing agents, forexample, may comprise a polymeric delivery vehicle. In exemplaryembodiments, the sustained release-enhancing agent may comprisepolyethylene glycol (PEG) coformulated or coordinately delivered withpeptide YY and one or more intranasal delivery-enhancing agents. PEG maybe covalently bound to peptide YY. The sustained release-enhancingmethods and formulations of the present invention will increaseresidence time (RT) of the peptide YY at a site of administration andwill maintain a basal level of the peptide YY over an extended period oftime in blood serum, CNS or gastrointestinal tract, or other tissue inthe mammalian subject.

Within other detailed embodiments of the invention, the foregoingmethods and formulations are administered to a mammalian subject toyield enhanced blood serum, gastrointestinal tract, or CNS levels, orother tissue levels of the peptide YY to maintain basal levels ofpeptide YY over an extended period of time. Exemplary methods andformulations involve administering a pharmaceutical formulationcomprising an intranasal effective amount of peptide YY and one or moreintranasal delivery-enhancing agents to a mucosal surface of thesubject, in combination with intramuscular or subcutaneousadministration of a second pharmaceutical formulation comprising peptideYY. Maintenance of basal levels of peptide YY is particularly useful fortreatment and prevention of disease, for example, obesity, cancer, ormalnutrition or wasting related to cancer.

The foregoing mucosal drug delivery formulations and preparative anddelivery methods of the invention provide improved mucosal delivery ofpeptide YY to mammalian subjects. These compositions and methods caninvolve combinatorial formulation or coordinate administration of one ormore peptide YY(s) with one or more mucosal (e.g., intranasal)delivery-enhancing agents. Among the mucosal delivery-enhancing agentsto be selected from to achieve these formulations and methods are (a)aggregation inhibitory agents; (b) charge modifying agents; (c) pHcontrol agents; (d) degradative enzyme inhibitors; (e) mucolytic ormucus clearing agents; (f) ciliostatic agents; (g) membranepenetration-enhancing agents (e.g. (i) a surfactant, (ii) a bile salt,(iii) a phospholipid or fatty acid additive, mixed micelle, liposome, orcarrier, (iv) an alcohol, (v) an enamine, (vi) an NO donor compound,(vii) a long-chain amphipathic molecule (viii) a small hydrophobicpenetration enhancer; (ix) sodium or a salicylic acid derivative; (x) aglycerol ester of acetoacetic acid, (xi) a cyclodextrin orbeta-cyclodextrin derivative, (xii) a medium-chain fatty acid, (xiii) achelating agent, (xiv) an amino acid or salt thereof, (xv) anN-acetylamino acid or salt thereof, (xvi) an enzyme degradative to aselected membrane component, (xvii) an inhibitor of fatty acidsynthesis, (xviii) an inhibitor of cholesterol synthesis; or (xix) anycombination of the membrane penetration enhancing agents of(i)-(xviii)); (h) modulatory agents of epithelial junction physiology,such as nitric oxide (NO) stimulators, chitosan, and chitosanderivatives; (i) vasodilator agents; (j) selective transport-enhancingagents; and (k) stabilizing delivery vehicles, carriers, supports orcomplex-forming species with which the peptide YY(s) is/are effectivelycombined, associated, contained, encapsulated or bound to stabilize theactive agent for enhanced nasal mucosal delivery.

In various embodiments of the invention, peptide YY is combined withone, two, three, four or more of the mucosal (e.g., intranasal)delivery-enhancing agents recited in (a)-(k), above. These mucosaldelivery-enhancing agents may be admixed, alone or together, withpeptide YY, or otherwise combined therewith in a pharmaceuticallyacceptable formulation or delivery vehicle. Formulation of peptide YYwith one or more of the mucosal delivery-enhancing agents according tothe teachings herein (optionally including any combination of two ormore mucosal delivery-enhancing agents selected from (a)-(k) above)provides for increased bioavailability of the peptide YY followingdelivery thereof to a mucosal (e.g., nasal mucosal) surface of amammalian subject.

In related aspects of the invention, a variety of coordinateadministration methods are provided for enhanced mucosal delivery ofpeptide YY. These methods comprise the step, or steps, of administeringto a mammalian subject a mucosally effective amount of at least onepeptide YY in a coordinate administration protocol with one or moremucosal delivery-enhancing agents selected from (a) aggregationinhibitory agents; (b) charge modifying agents; (c) pH control agents;(d) degradative enzyme inhibitors; (e) mucolytic or mucus clearingagents; (f) ciliostatic agents; (g) membrane penetration-enhancingagents (e.g. (i) a surfactant, (ii) a bile salt, (iii) a phospholipid orfatty acid additive, mixed micelle, liposome, or carrier, (iv) analcohol, (v) an enamine, (vi) an NO donor compound, (vii) a long-chainamphipathic molecule (viii) a small hydrophobic penetration enhancer;(ix) sodium or a salicylic acid derivative; (x) a glycerol ester ofacetoacetic acid, (xi) a cyclodextrin or beta-cyclodextrin derivative,(xii) a medium-chain fatty acid, (xiii) a chelating agent, (xiv) anamino acid or salt thereof, (xv) an N-acetylamino acid or salt thereof,(xvi) an enzyme degradative to a selected membrane component, (xvii) aninhibitor of fatty acid synthesis, (xviii) an inhibitor of cholesterolsynthesis; or (xix) any combination of the membrane penetrationenhancing agents of (i)-(xviii)); (h) modulatory agents of epithelialjunction physiology, such as nitric oxide (NO) stimulators, chitosan,and chitosan derivatives; (i) vasodilator agents; (j) selectivetransport-enhancing agents; and (k) stabilizing delivery vehicles,carriers, supports or complex-forming species with which the peptideYY(s) is/are effectively combined, associated, contained, encapsulatedor bound to stabilize the active agent for enhanced mucosal (e.g.,intranasal) delivery.

To practice a coordinate administration method according to theinvention, any combination of one, two or more of the mucosaldelivery-enhancing agents recited in (a)-(k), above, may be admixed orotherwise combined for simultaneous mucosal (e.g., intranasal)administration. Alternatively, any combination of one, two or more ofthe mucosal delivery-enhancing agents recited in (a)-(k) can bemucosally administered, collectively or individually, in a predeterminedtemporal sequence separated from mucosal administration of the peptideYY (e.g., by pre-administering one or more of the delivery-enhancingagent(s)), and via the same or different delivery route as the peptideYY (e.g., to the same or to a different mucosal surface as the peptideYY, or even via a non-mucosal (e.g., intramuscular, subcutaneous, orintravenous route). Coordinate administration of peptide YY with anyone, two or more of the mucosal delivery-enhancing agents according tothe teachings herein provides for increased bioavailability of thepeptide YY following delivery thereof to a mucosal surface of amammalian subject.

In additional related aspects of the invention, various“multi-processing” or “co-processing” methods are provided for preparingformulations of peptide YY for enhanced nasal mucosal delivery. Thesemethods comprise one or more processing or formulation steps wherein oneor more peptide YY(s) is/are serially, or simultaneously, contactedwith, reacted with, or formulated with, one, two or more (including anycombination of) of the mucosal delivery-enhancing agents as detailedabove. To practice the multi-processing or co-processing methodsaccording to the invention, the peptide YY is/are exposed to, reactedwith, or combinatorially formulated with any combination of one, two ormore of the mucosal delivery-enhancing agents recited in (a)-(k), above,either in a series of processing or formulation steps, or in asimultaneous formulation procedure, that modifies the peptide YY (orother formulation ingredient) in one or more structural or functionalaspects, or otherwise enhances mucosal delivery of the active agent inone or more (including multiple, independent) aspect(s) that are eachattributed, at least in part, to the contact, modifying action, orpresence in a combinatorial formulation, of a specific mucosaldelivery-enhancing agent recited in (a)-(k), above.

In certain detailed aspects of the invention, the methods andcompositions which comprise a mucosally effective amount of peptide YYand one or more mucosal delivery-enhancing agent(s) (combined in apharmaceutical formulation together or administered in a coordinatenasal mucosal delivery protocol) provide nasal transmucosal delivery ofthe peptide YY in a pulsatile delivery mode to maintain more consistentor normalized, and/or elevated levels of peptide YY in the blood serum.In this context, the pulsatile delivery methods and compositions of theinvention yield increased bioavailability (e.g., as measured by maximalconcentration, (C_(max)) or area under concentration curve (AUC) ofpeptide YY and/or an increased mucosal delivery rate (e.g., as measuredby time to maximal concentration (t_(max)), C_(max) and/or AUC comparedto other mucosal or non-mucosal delivery method-based controls. Forexample, the invention provides pulsatile delivery methods andformulations comprise peptide YY and one or more mucosaldelivery-enhancing agent(s), wherein the formulation administeredmucosally (e.g., intranasally) to a mammalian subject, yields an areaunder the concentration curve (AUC) for peptide YY in the blood serum,CNS or gastrointestinal tract that is about 10% or greater compared toan area under the concentration curve (AUC) for peptide YY in the bloodserum, CNS or gastrointestinal tract following subcutaneous injection tothe mammalian subject.

Often the formulations of the invention are administered to a nasalmucosal surface of the subject. In certain embodiments, the peptide YYis a human peptide YY, for example, Peptide YY (human: (SEQ ID NO: 792)H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂);(Leu³¹,Pro³⁴)-Peptide YY (human): (SEQ ID NO: 798)H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Pro-Arg-Tyr-NH₂;(Pro³⁴)-PYY (human: (SEQ ID NO: 799)H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Pro-Arg-Tyr-NH₂);PYY₃₋₃₆ (human: (SEQ ID NO: 794)H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂);Peptide YY₁₃₋₃₆ (porcine, rat: (SEQ ID NO: 793)H-Ser-Pro-Glu-Glu-Leu-Ser-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂);(Bachem AG, King of Prussia, Pa., United States). The PYY peptide hasbeen isolated from human colonic extracts. It is identical in sequenceto porcine PYY except for two amino acid replacements. See Tatemoto, etal., Biochem. Biophys. Res. Commun. 157:713, 1988, incorporated hereinby reference. PYY₃₋₃₆ form of PYY was found in human intestine andcirculating blood. In contrast to PYY, PYY₃₋₃₆ selectively binds to Y₂receptors. Peptide YY₁₃₋₃₆, the C-terminal fragment, was shown tosuppress the noradrenaline release from sympathetic nerve endings. Itthereby mimics the effects of PYY and NPY at presynaptic (Y₂) receptors.The peptide was also able to compete with NPY for essentially allbinding sites in rat brain. A mucosally effective dose of peptide YYwithin the pharmaceutical formulations of the present inventioncomprises, for example, between about 0.001 pmol to about 100 pmol perkg body weight, between about 0.01 pmol to about 10 pmol per kg bodyweight, or between about 0.1 pmol to about 5 pmol per kg body weight. Infurther exemplary embodiments, dosage of peptide YY is between about 0.5pmol to about 1.0 pmol per kg body weight. The pharmaceuticalformulations of the present invention may be administered one or moretimes per day (for example, before a meal), or 3 times per week or onceper week for between one week and 96 weeks. In certain embodiments, thepharmaceutical formulations of the invention is administered one or moretimes daily, two times daily, four times daily, six times daily, oreight times daily. In related embodiments, the mucosal (e.g.,intranasal) formulations comprising peptide YY(s) and one or moredelivery-enhancing agent(s) administered via a repeated dosing regimenyields an area under the concentration curve (AUC) for peptide YY in theblood plasma or CSF following repeated dosing that is about 10% orgreater compared to an area under the concentration curve (AUC) forpeptide YY in the plasma or CSF following one or more subcutaneousinjections of the same or comparable amount of peptide YY. In otherembodiments, the mucosal formulations of the invention administered viaa repeated dosing regimen yields an area under the concentration curve(AUC) for peptide YY in the blood plasma or CNS following repeateddosing that is about 25% or greater, or about 40%, 80%, 100%, 150%, orgreater, compared to the AUC for peptide YY in the blood plasma or CNSfollowing one or more subcutaneous injections of the same or comparableamount of peptide YY.

In a further embodiment, the mucosal (e.g., intranasal) formulations ofthe present invention comprising peptide YY(s) and one or moredelivery-enhancing agent(s) administered via a dosing regimen yield anarea under the concentration curve (AUC) for peptide YY in the bloodplasma or CSF that is about 110% or greater compared to an area underthe concentration curve (AUC) for peptide YY in the plasma or CSFfollowing mucosal delivery of peptide YY in water or saline withoutenhancers of the same or comparable amount of peptide YY to a mammaliansubject. In other embodiments, the mucosal formulations of the inventionadministered via a dosing regimen yields an area under the concentrationcurve (AUC) for peptide YY in the blood plasma or CNS that is about 125%or greater, or about 140%, 180%, 200%, 250%, or greater, compared to theAUC for peptide YY in the blood plasma or CNS following mucosal deliveryof peptide YY in water or saline without enhancers of the same orcomparable amount of peptide YY to a mammalian subject. In a moredetailed embodiment, the dosing regimen is, for example, a single dosingregimen or a repeated dosing regimen.

Often the formulations of the invention are administered to a nasalmucosal surface of the subject. In certain embodiments, the formulationcomprises neuropeptide Y or pancreatic peptide, having peptide YY-likeactivity, for example, neuropeptide Y (human, rat),([¹²⁵I]-Tyr)-neuropeptide Y (human, rat), biotinyl-neuropeptide Y(human, rat), neuropeptide Y (free acid) (human, rat),(Leu³¹,Pro³⁴)-neuropeptide Y (human, rat), (D-Trp³²)-neuropeptide Y(human, rat), (Tyr(Me)²¹)-neuropeptide Y (human, rat), neuropeptide Y(porcine), (Ala³¹,Aib³²)-neuropeptide Y (porcine),(Leu³¹,Pro³⁴)-neuropeptide Y (porcine), (Pro³⁴)-neuropeptide Y(porcine), (D-Trp³²)-neuropeptide Y (porcine), neuropeptide Y₁₋₂₄ amide(human, rat), (Cys²)-neuropeptideY₁₋₄-8-aminooctanoyl-(D-Cys²⁷)-neuropeptide Y₂₅₋₃₂, neuropeptide Y₂₋₃₆(human, rat), neuropeptide Y₂₋₃₆ (porcine), neuropeptide Y₃₋₃₆ (human,rat), neuropeptide Y₃₋₃₆ (porcine), neuropeptide Y₁₃₋₃₆ (human, rat),(Leu³¹,Pro³⁴)-neuropeptide Y₁₃₋₃₆ (human, rat), neuropeptide Y₁₃₋₃₆(porcine), neuropeptide Y₁₈₋₃₆, pancreaticpolypeptide₁₋₁₇-(Ala³¹,Aib³²)-neuropeptide Y₁₈₋₃₆ (human), neuropeptideY₂₂₋₃₆, Tyr-Lys-Gly-Arg-(Glu²⁶,Lys²⁹,Pro³⁴)-neuropeptide Y₂₆₋₃₆,(D-Tyr^(27,36),D-Thr³²)-neuropeptide Y₂₇₋₃₆, ((Cys³¹,Nva³⁴)-neuropeptideY₂₇₋₃₆)₂, (Pro³⁰,Tyr³²,Leu³⁴)-neuropeptide Y₂₈₋₃₆,(His³²,Leu³⁴)-neuropeptide Y₃₂₋₃₆, ([¹²⁵I]-Tyr)-neuropeptide Y(porcine), ([¹²⁵I]-Tyr)-(Leu³¹,Pro³⁴)-neuropeptide Y (porcine),(Gly¹,Ser^(3,22),Gln^(4,34),Thr⁶,Ala¹⁹,Tyr²¹,Ala^(23,31),Aib³²)-pancreaticpolypeptide (human).

In certain detailed aspects of the invention, a stable pharmaceuticalformulation is provided which comprises peptide YY and one or moredelivery-enhancing agent(s), wherein the formulation administeredintranasally to a mammalian subject yields a time to maximal plasmaconcentration (t_(max)) for peptide YY between approximately 0.4 to 2.0hours in a mammalian subject. Often the formulation is administered to anasal mucosal surface of the subject.

In certain embodiments of the invention, the intranasal formulation ofpeptide YY and one or more delivery-enhancing agent(s) yields a time tomaximal plasma concentration (t_(max)) for peptide YY betweenapproximately 0.4 to 1.5 hours in the mammalian subject. Alternately,the intranasal formulation of the present invention yields a time tomaximal plasma concentration (t_(max)) for peptide YY betweenapproximately 0.7 to 1.5 hours, or between approximately 1.0 to 1.3hours in the mammalian subject.

In certain detailed aspects of the invention, a stable pharmaceuticalformulation is provided which comprises peptide YY and one or moreintranasal delivery-enhancing agent(s), wherein the formulationadministered intranasally to a mammalian subject yields a peakconcentration of peptide YY in the blood plasma (C_(max)) or CNSfollowing intranasal administration to the subject by methods andcompositions of the present invention is about 10% or greater comparedto a peak concentration of peptide YY in the blood plasma or CNSfollowing subcutaneous injection to the mammalian subject. Withinrelated methods, the formulation is administered to a nasal mucosalsurface of the subject.

In other detailed embodiments of the invention, the intranasalformulation of the peptide YY(s) and one or more delivery-enhancingagent(s) yields a peak concentration of peptide YY in blood plasma(C_(max)) or CNS following intranasal administration to the subject thatis about 25% or greater compared to a peak concentration of peptide YYin the blood plasma or CNS following subcutaneous injection of acomparable dose of peptide YY to the subject. Alternately, theintranasal formulation of the present invention may yield a peakconcentration of peptide YY in the blood plasma (C_(max)) or CNS that isabout 40%, 80%, 100% or 150%, or greater compared to the peakconcentration of peptide YY in the blood plasma or CNS followingsubcutaneous injection to the mammalian subject.

In a further embodiment, the mucosal (e.g., intranasal) formulations ofthe present invention of the peptide YY(s) and one or moredelivery-enhancing agent(s) administered via a dosing regimen yield apeak concentration of peptide YY in blood plasma (C_(max)) or CNSfollowing intranasal administration to the subject that is about 110% orgreater compared to a peak concentration of peptide YY in the bloodplasma or CNS following mucosal delivery of peptide YY in water orsaline without enhancers of the same or comparable amount of peptide YYto a mammalian subject. In other embodiments, the mucosal formulationsof the present invention administered via a dosing regimen yield a peakconcentration of peptide YY in the blood plasma (C_(max)) or CNS that isabout 125%, 140%, 180%, 200% or 250%, or greater compared to the peakconcentration of peptide YY in the blood plasma or CNS following mucosaldelivery of peptide YY in water or saline without enhancers of the sameor comparable amount of peptide YY to a mammalian subject. In a moredetailed embodiment, the dosing regimen is, for example, a single dosingregimen or a repeated dosing regimen.

Intranasal delivery-enhancing agents are employed which enhance deliveryof peptide YY into or across a nasal mucosal surface. For passivelyabsorbed drugs, the relative contribution of paracellular andtranscellular pathways to drug transport depends upon the pKa, partitioncoefficient, molecular radius and charge of the drug, the pH of theluminal environment in which the drug is delivered, and the area of theabsorbing surface. The intranasal delivery-enhancing agent of thepresent invention may be a pH control agent. The pH of thepharmaceutical formulation of the present invention is a factoraffecting absorption of peptide YY via paracellular and transcellularpathways to drug transport. In one embodiment, the pharmaceuticalformulation of the present invention is pH adjusted to between about pH3.0 to 6.0. In a further embodiment, the pharmaceutical formulation ofthe present invention is pH adjusted to between about pH 3.0 to 5.0. Ina further embodiment, the pharmaceutical formulation of the presentinvention is pH adjusted to between about pH 4.0 to 5.0. In a furtherembodiment, the pharmaceutical formulation of the present invention ispH adjusted to about pH 5.0±0.5.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides improved methods andcompositions for nasal mucosal delivery of peptide YY to mammaliansubjects for treatment or prevention of a variety of diseases andconditions. Examples of appropriate mammalian subjects for treatment andprophylaxis according to the methods of the invention include, but arenot restricted to, humans and non-human primates, livestock species,such as horses, cattle, sheep, and goats, and research and domesticspecies, including dogs, cats, mice, rats, guinea pigs, and rabbits.

In order to provide better understanding of the present invention, thefollowing definitions are provided:

Peptide YY

The term, “pancreatic polypeptide family,” as used herein, is comprisedof three naturally occurring bioactive peptides, “pancreatic polypeptide(PP)”, “neuropeptide Y (NPY),” and “peptide YY (PYY),” that are found inthe gastrointestinal tract, blood serum, and brain. PYY is released fromendocrine L cells of the distal digestive tract by indirect stimulationfrom the proximal gut through neural and humoral pathways and by directstimulation of L cells by luminal contents. Two endogenous forms ofpeptide YY, PYY₁₋₃₆ and PYY₃₋₃₆, are released into the circulation by ameal. Proposed gastrointestinal actions of PYY are inhibition of gastricsecretion, inhibition of pancreatic secretion inhibition of intestinalsecretion, and inhibition of gastrointestinal motility.

As used herein, “peptide YY” refers to “peptide YY (PYY)” innative-sequence or in variant form, as well as derivatives, fragments,and analogs of peptide YY, and from any source, whether natural,synthetic, or recombinant. Examples include human peptide YY (PYY),which is natural or recombinant peptide YY with the human nativesequence, and recombinant peptide YY. U.S. Pat. Nos. 5,604,203 and5,574,010, each incorporated herein by reference. The term “peptide YY”as used herein, is intended to include recombinant or natural humanpeptide YY.

As used herein, “pancreatic polypeptide (PP)” or “neuropeptide Y (NPY)”has peptide YY-like activity, or may be a peptide YY agonist binding toa “Y receptor.” In certain formulations, “pancreatic polypeptide (PP)”or “neuropeptide Y (NPY)” may be substituted for peptide YY.

The term, “Y receptor,” as used herein, refers to PYY binding andactivating at least three receptor sub types (Y₁, Y₂, and Y₅) in ratsand humans. PYY may interact with a postulated fourth subtype, theperipheral Y₂-like receptor. The nomenclature for Y receptors suggestedby the International Union of Pharmacology is used. Bjork, et al.,Scand. J. Gastroenterol. 28:879-884, 1993, incorporated herein byreference. These Y receptor subtypes display different patterns ofaffinity and activation for the two endogenous ligands PYY and PYY₃₋₃₆and for two synthetic analogs, for example, [Pro³⁴]PYY and [D-Trp³²]PYY.In general, the Y₁ receptor subtype has high affinity for PYY and[Pro³⁴]PYY, the Y₂ has high affinity for PYY and PYY₃₋₃₆, and the Y₅subtype binds PYY, PYY₃₋₃₆, [Pro³⁴]PYY, and [D-Trp³²]PYY with highaffinity. These different patterns of receptor selectivity could becaused by differences in primary structure, differences in tertiarystructure due to altered conformations of the ligands in solution, orboth. Neuropeptide Y1 and Y5 receptor gene variant polymorphisms areassociated with reduced serum triglyceride level and elevated HDLcholesterol in severely obese mammalian subjects. Neuropeptide Y1 and Y5receptor gene variants are likely a gain-in-function polymorphism.Blumenthal, et al., Clin. Genet. 62:196-202, 2002, incorporated hereinby reference.

The term, “Y receptor antagonist”, as used herein, refers to a smallmolecule, for example dihydropyridine or a peptide, for example, apeptide YY analog, that binds to the Y receptor and inhibits the normalphysiological activity of peptide YY, neuropeptide Y, or pancreaticpeptide. Poindexter, et al., Bioorganic & Medicinal Chemistry Letters12:379-382, 2002, incorporated herein by reference. Methods of treatinga feeding disorder may require administration of a compound that is a Y5receptor antagonist effective to inhibit the subject's Y5 receptor. U.S.Application No. 20020103123, incorporated herein by reference.

The term, “peptide YY agonist” or “Y receptor agonist,” as used herein,refers to any compound which elicits an effect of peptide YY (PYY) toreduce nutrient availability leading to weight loss (e.g., PYY₃₋₃₆), orincrease nutrient availability leading to weight gain (e.g., PYY₁₋₃₆),for example, a compound (1) having activity in the food intake, gastricemptying, pancreatic secretion, or weight loss assays, and (2) whichbinds specifically in a Y receptor assay or in a competitive bindingassay with labeled PYY or PYY₃₋₃₆ from certain tissues having anabundance of Y receptors, including e.g., area postrema, wherein the PYYagonist is not pancreatic polypeptide. Preferably, PYY agonists wouldbind in such assays with an affinity of greater than 1 μM, and morepreferably with an affinity of greater than 1-5 nM. “Peptide YY agonist”or “Y receptor agonist”, as used herein, refers to a small molecule or apeptide, for example, peptide YY, neuropeptide Y, pancreatic peptide, ora peptide YY analog, that binds to the Y receptor and produces thenormal physiological activity of peptide YY, neuropeptide Y, orpancreatic peptide.

The term, “peptide YY agonist” or “Y receptor agonist,” as used herein,may be a fusion protein containing the PYY peptide sequence or may beformed by modifying the PYY peptide's natural amino acid sequence ormodifying the N-terminal amino and/or the C-terminal carboxyl group, andinclude salts formed with acids and/or bases, particularlyphysiologically acceptable inorganic and organic acids and bases.Preferred modifications are those which provide a more stable, activepeptide which will be less prone to enzymatic degradation in vivo.

In certain embodiments, the peptide YY is a human peptide YY, forexample, Peptide YY (human: (SEQ ID NO: 792)H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂);(Leu³¹,Pro³⁴)-Peptide YY (human: (SEQ ID NO: 798)H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Pro-Arg-Tyr-NH₂);(Pro³⁴)-Peptide YY (human: (SEQ ID NO: 799)H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Pro-Arg-Tyr-NH₂);Peptide YY₃₋₃₆ (human: (SEQ ID NO: 794)H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂);Peptide Y₁₃₋₃₆ (porcine, rat: (SEQ ID NO: 793)H-Ser-Pro-Glu-Glu-Leu-Ser-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂);(Bachem AG, King of Prussia, Pa., United States).

The term “endogenous ligand PYY,” as used herein, refers to, forexample, PYY, a 36 amino acid endogenous polypeptide, and PYY₃₋₃₆, atruncated endogenous polypeptide.

The term “synthetic analogs of PYY,” as used herein, refers topolypeptides with natural or synthetic amino acid substitutionsreplacing the endogenous ligand PYY, for example, [Pro³⁴]PYY and[D-Trp³²]PYY. Synthetic analogs of PYY have biological activity that iscomparable or higher than the biological activity of endogenous ligandPYY. See U.S. Pat. Nos. 5,604,203 and 5,574,010, each incorporatedherein by reference.

Peptide YY (PYY) is a 36 amino acid residue peptide amide isolatedoriginally from porcine intestine and localized in the endocrine cellsof the gastrointestinal tract and the pancreas. Tatemotu, et al., Proc.Natl. Acad. Sci. 79:2514, 1982, incorporated herein by reference. Theamino acid sequences of porcine and human PYY are as follows: porcinePYY-YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY (SEQ ID NO: 791); humanPYY-YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 792). The aminoacid sequence for canine and rat PYY is the same as porcine PYY. PYY isbelieved to inhibit gut motility and blood flow, to mediate intestinalsecretion, and stimulate net absorption. Laburthe, Trends Endocrinol.Metab. 1:168, 1990; Cox, et al., Br. J. Pharmacol. 101:247, 1990;Playford, et al., Cancer 335:1555, 1990; MacFayden, et al.,Neuropeptides 7:219, 1986, each incorporated herein by reference. Novelanalogs have been prepared in order to emulate and preferably enhancethe duration of effect, biological activity, and selectivity of thenatural peptide. Many of these analogs are derived from biologicallyactive peptide fragments of PYY (e.g., PYY₂₂₋₃₆ and PYY₂₅₋₃₆). Suchanalogs, which inhibit the proliferation of pancreatic tumors, arepeptide YY agonists. U.S. Pat. No. 5,574,010, incorporated herein byreference.

Additional peptide YY agonists which can be used to practice thetherapeutic method of the present invention include, but are not limitedto, those specifically recited in the publications set forth below: U.S.Pat. Nos. 5,604,203 and 5,574,010; Balasubramaniam, et al., PeptideResearch 1:32, 1988; Japanese Patent Application No. 2,225,497, 1990;Balasubramaniam, et al., Peptides 14:1011, 1993; Grandt, et al., Reg.Peptides 51:151, 1994; and PCT International Application No. 94/03380,each incorporated herein by reference.

Peptide YY agonists which can be used to practice the therapeutic methodof the present invention also include the closely related peptideneuropeptide Y (NPY) as well as derivatives, fragments, and analogs ofNPY. The amino acid sequences of porcine and human NPY are as follows:human neuropeptide Y: YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY (SEQ ID NO:795); porcine neuropeptide Y: YPSKPDNPGEDAPAEDLARYYSALRHYINLITRQRY (SEQID NO: 796). The amino acid sequence for rat neuropeptide Y, rabbitneuropeptide Y, and guinea pig neuropeptide Y are the same as humanneuropeptide Y. See U.S. Pat. Nos. 5,604,203 and 5,574,010, eachincorporated herein by reference.

Often the formulations of the invention are administered to a nasalmucosal surface of the subject. In certain embodiments, “pancreaticpolypeptide (PP)” or “neuropeptide Y (NPY)” has peptide YY-likeactivity. In certain formulations, “pancreatic polypeptide (PP)” or“neuropeptide Y (NPY)” may be substituted for peptide YY, for example,neuropeptide Y (human, rat), ([¹²⁵I]-Tyr)-neuropeptide Y (human, rat),biotinyl-neuropeptide Y (human, rat), neuropeptide Y (free acid) (human,rat), (Leu³¹,Pro³⁴)-neuropeptide Y (human, rat), (D-Trp³²)-neuropeptideY (human, rat), (Tyr(Me)²¹)-neuropeptide Y (human, rat), neuropeptide Y(porcine), (Ala³¹,Aib³²)-neuropeptide Y (porcine),(Leu³¹,Pro³⁴)-neuropeptide Y (porcine), (Pro³⁴)-neuropeptide Y(porcine), (D-Trp³²)-neuropeptide Y (porcine), neuropeptide Y₁₋₂₄ amide(human, rat), (Cys²)-neuropeptideY₁₋₄-8-aminooctanoyl-(D-Cys²⁷)-neuropeptide Y₂₅₋₃₂, neuropeptide Y₂₋₃₆(human, rat), neuropeptide Y₂₋₃₆ (porcine), neuropeptide Y₃₋₃₆ (human,rat), neuropeptide Y₃₋₃₆ (porcine), neuropeptide Y₁₃₋₃₆ (human, rat),(Leu³¹,Pro³⁴)-neuropeptide Y₁₃₋₃₆ (human, rat), neuropeptide Y₁₃₋₃₆(porcine), neuropeptide Y₁₈₋₃₆, pancreaticpolypeptide₁₋₁₇-(Ala³¹,Aib³²)-neuropeptide Y₁₈₋₃₆ (human), neuropeptideY₂₂₋₃₆, (SEQ ID NO: 797)Tyr-Lys-Gly-Arg-(Glu²⁶,Lys²⁹,Pro³⁴)-neuropeptide Y₂₆₋₃₆, (D-Tyr^(27,36)D-Thr³²)-neuropeptide Y₂₇₋₃₆, ((Cys³¹,Nva³⁴)-neuropeptide Y₂₇₋₃₆)₂,(Pro³⁰,Tyr³²,Leu³⁴)-neuropeptide Y₂₈₋₃₆, (His³²,Leu³⁴)-neuropeptideY₃₂₋₃₆, ([¹²⁵I]-Tyr)-neuropeptide Y (porcine),([¹²⁵I]-Tyr)-(Leu³¹,Pro³⁴)-neuropeptide Y (porcine),(Gly¹,Ser^(3,22),Gln^(4,34),Thr⁶,Ala¹⁹,Tyr²¹,Ala^(23,31),Aib³²)-pancreaticpolypeptide (human).

Examples of neuropeptide Y analogs include but are not limited to, thosespecifically recited in the publications set forth below. See U.S. Pat.Nos. 5,604,203 and 5,574,010; German Patent Application No. DE 3811193A1(1989); Balasubramaniam, et al., J. Biological Chem. 265:14724-14727,1990; Cox, et al., Br. J. Pharmacol. 101:247-252, 1990; PCT ApplicationNo. WO 91/08223, 1991; U.S. Pat. No. 5,026,685, 1991; Balasubramaniam,et al., J. Biological Chem. 267:4680-4685, 1992; European PatentApplication No. 0355793 A3, 1992; Dumont, et al., European J. Pharmacol.238:37-45, 1993; Kirby, et al., J. Med. Chem. 36:3802-3808, 1993; PCTApplication No. WO 94/00486, 1994; Fournier, et al., MolecularPharmacol. 45:93-101, 1994; Balasubramaniam, et al., J. Med. Chem.37:811-815, 1994; Polter, et al., European J. Pharmacol. 267:253-262,1994; and U.S. Pat. No. 5,328,899, each incorporated herein byreference.

Early descriptions of the structure of avian PP were derived fromanalysis of X-ray crystallography data which led to modeling ofpotential structures of mammalian PP, NPY, and PYY by computer analysis.The solution structures of PP and of NPY have been studied by circulardichroism (CD) and nuclear magnetic resonance (NMR). All analyses of PPhave consistently found evidence for folded structure (the “PP fold”)stabilized by hydrophobic interactions among residues in the NH₂- andCOOH-terminal portions. A similar structure has been assumed to existfor NPY and PYY because of their high sequence homologies to PP. Suchresults have led to hypotheses that this stable structure of PP familypeptides is critical for binding and activation of PP/NPY/PYY-specificreceptors and that receptor selectivity depends in part on differencesin solution structure produced by amino acid deletions or substitutionsin naturally occurring or synthetic Y receptor agonists.

Further examples of peptide YY, neuropeptide Y, pancreatic polypeptide,neuropeptide Y receptors: Y1, Y2, Y3, Y4, and Y5; neuropeptide Yreceptor agonists, and neuropeptide Y antagonists may be found, forexample, in U.S. Pat. Nos. 6,368,824; 6,355,478; 6,207,799; 6,075,009;5,989,920; 5,989,834; 5,976,814; 5,968,819; 5,965,392; 5,696,093;5,670,482; 5,602,024; 5,621,079; or 5,571,695, each incorporated hereinby reference.

Peptides and proteins used in the methods and compositions of theinvention can be obtained by a variety of means. Many peptides andproteins can be readily obtained in purified form from commercialsources. Smaller peptides (less than 100 amino acids long) can beconveniently synthesized by standard chemical methods familiar to thoseskilled in the art. Creighton, Proteins: Structures and MolecularPrinciples, W.H. Freeman and Co., N.Y., 1983, incorporated herein byreference. Larger peptides (longer than 100 amino acids) can be producedby a number of methods including recombinant DNA technology. See, forexample, the techniques described in Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y., 1989; andAusubel et al., eds., Current Protocols in Molecular Biology, GreenPublishing Associates, Inc., and John Wiley & Sons, Inc., N.Y, 1989,each incorporated herein by reference. Alternatively, RNA encoding theproteins can be chemically synthesized. See, for example, the techniquesdescribed in Oligonucleotide Synthesis, Gait, M. J., ed., IRL Press,Oxford, 1984, incorporated herein by reference.

Mucosal Delivery Enhancing Agents

“Mucosal delivery enhancing agents” or “intranasal delivery-enhancingagent(s)” are defined as chemicals and other excipients that, when addedto a formulation comprising water and peptide YY (the controlformulation) produce a formulation that produces a significant increasein transport of peptide YY across the mucosa or nasal mucosa,respectively, as measured by the maximum blood, serum, or cerebralspinal fluid concentration (C_(max)) or by the area under the curve,AUC, in a plot of concentration versus time.

Treatment and Prevention of Obesity

As noted above, the instant invention provides improved and usefulmethods and compositions for nasal mucosal delivery of one or morepeptide YY compound(s) to prevent and treat obesity in mammaliansubjects. As used herein, prevention and treatment of obesity meanprevention of the onset or lowering the incidence or severity ofclinical obesity by reducing food intake during meals. In certainaspects, the pharmaceutical formulations and methods of the inventionprevent or alleviate obesity in mammalian subjects. In certainembodiments, the one or more peptide YY compound(s) may be a peptide YYanalog, for example, peptide YY₃₋₃₆.

Obesity is a recognized risk factor for hypertension, arteriosclerosis,congestive heart failure, stroke, gallbladder disease, osteoarthritis,sleep apnea, reproductive disorders such as polycystic ovarian syndrome,cancers of the breast, prostate, and colon, and increased incidence ofcomplications of general anesthesia. Kopelman, Nature 404:635-43, 2000,incorporated herein by reference. It reduces life-span and carries aserious risk of co-morbidities above, as well disorders such asinfections, varicose veins, acanthosis nigricans, eczema, exerciseintolerance, insulin resistance, hypertension hypercholesterolemia,cholelithiasis, orthopedic injury, and thromboembolic disease. Rissanen,et al., BMJ 301:835-7, 1990, incorporated herein by reference. Obesityis also a risk factor for the group of conditions called insulinresistance syndrome, or “Syndrome X.”

The instant invention provides improved and useful methods andcompositions for nasal mucosal delivery of peptide YY to regions of thebrain, for example, the hypothalamus or the proopiomelanocortin (POMC)and NPY arcuate neurons, to prevent and treat obesity in mammaliansubjects. Experimental results support the hypothesis that the cells inthe arcuate nucleus detect circulating peripheral satiety signals andrelay these signals to other brain regions. This is further supported bythe observation that leptin modifies the activity of both theproopiomelanocortin (POMC) and NPY arcuate neurons. Experiments haveshown, through a combination of electrophysiological and hypothalamicexplant studies, that the gut hormone, PYY₃₋₃₆, can directly influencehypothalamic circuits, which results in coordinate changes in POMC andNPY action. In addition, PYY₃₋₃₆ administered directly into this brainregion reduces food intake. Data show that postprandial concentrationsof PYY₃₋₃₆ inhibit food intake in both rodents and man for up to 12 h,which suggests that PYY₃₋₃₆ has a role in ‘longer term’ regulation offood intake. This contrasts with previously characterized gut-derived‘short-term’ satiety signals such as cholecystokinin, the effects ofwhich are relatively short-lived. The failure of PYY₃₋₃₆ to inhibit foodintake in Y2r-null mice provides further evidence that PYY₃₋₃₆ reducesfood intake through a Y2R-dependent mechanism. Experimental resultssuggest that a gut-hypothalamic pathway that involves postprandialPYY₃₋₃₆ acting at the arcuate Y2R has a role in regulating feeding.Thus, the PYY₃₋₃₆ system provides a therapeutic target for the treatmentof obesity. Nature 418:650-654, 2002, incorporated herein by reference.

The instant invention provides improved and useful methods andcompositions for nasal mucosal delivery of Y1 receptor antagonists toprevent and treat obesity in mammalian subjects. Y1 receptor antagonistsmay counter the effect of neuropeptide Y (NPY) on brain stem and centralnervous system neurophysiological responses to gastric loads. Y1receptor antagonists are, for example, dihydropyridine. Poindexter, etal., Bioorganic & Medicinal Chemistry Letters 12:379-382, 2002,incorporated herein by reference. Leptin is an adiposity hormone thatmodulates the activity of multiple hypothalamic signaling pathwaysinvolved in the control of food intake. Experiments demonstrate thatcentral forebrain administration of two feeding modulatory peptides,leptin and NPY, have opposing effects on brain stem; leptin increasesthe neurophysiological response to ascending meal-related feedbacksignals in the nucleus of the solitary tract (NTS), and NPY reduces it.The data are consistent with the suggestion that leptin reduces foodintake by enhancing the salience of gut negative feedback signals duringa meal, whereas NPY may in part increase intake by attenuating thefeeding inhibitory potency of these signals. These modulatory effects donot appear to be due to the peptides' downstream effects ongastrointestinal motility or on their ability to directly stimulateprimary vagal afferent input to the NTS, because gut vagal afferentresponses to the same range of gastric loads were unchanged by centraladministration of either peptide. Experiments were designed to evaluatewhether central administration of leptin or one of its downstreammediators, neuropeptide Y (NPY), could affect food intake by modulatingthe brain stem neurophysiological response to ascending meal-relatedfeedback signals in the nucleus of the solitary tract (NTS) inanesthetized male Long-Evans rats. NTS neurons at the rostrocaudal levelof the area postrema were dose-dependently activated by gastric loadsranging from 2-10 ml, and leptin and NPY had opposite modulatory effectson this load volume/activity relationship: leptin significantlyincreased NTS responses to gastric loads, whereas NPY reduced thepotency and efficacy with which gastric loads activated NTS neurons.These effects were probably not mediated by peripheral effects ofcentrally administered peptides or by the gastrokinetic effects ofcentral NPY or leptin, because the dose-response relationship betweengastric load volume and neurophysiological firing rate was unchanged ingastric load-sensitive vagal afferent fibers. These data suggest amechanistic framework for considering how feeding behavior occurring inmeals is altered by challenges to energy homeostasis, such as fastingand overfeeding. Schwartz, et al., Endocrinology 143:3779-3784, 2002,incorporated herein by reference. Nasal mucosal delivery of Y1 receptorantagonists of the present invention are useful to prevent and treatobesity in mammalian subjects. Y1 receptor antagonists may counter theeffect of neuropeptide Y (NPY) on brain stem and central nervous systemneurophysiological responses to gastric loads.

Treatment and Prevention of Alzheimer's Disease

As noted above, the instant invention provides improved and usefulmethods and compositions for nasal mucosal delivery of one or morepeptide YY compound(s) to prevent and treat Alzheimer's disease inmammalian subjects. It is proposed that aluminum is involved in theinitiation and progression of Alzheimer's disease. Young patients withAlzheimer's disease are reported to absorb more aluminum from theintestinal tract than unafflicted controls. Similarly, persons with Downsyndrome, a population at high risk for developing Alzheimer's disease,have been reported to absorb orally administered aluminum at anincreased rate compared to diploid controls. Experimental administrationof supraphysiological doses of human recombinant PYY to both diploid andpartially trisomic, Ts65Dn, mice dramatically decreases brain aluminumconcentrations suggests the possibility that chelation of aluminum bythis peptide may provide new insights into its role and that of aluminumin Alzheimer's disease. J. Inorg. Biochem. 87:51-56, 2001, incorporatedherein by reference. In certain embodiments, the one or more peptide YYcompound(s) may be a peptide YY analog, agonist or antagonist compound.

Treatment and Prevention of Colon Adenocarcinoma

As noted above, the instant invention provides improved and usefulmethods and compositions for nasal mucosal delivery of one or morepeptide YY compound(s) to prevent and treat colon carcinoma and colonadenocarcinoma in mammalian subjects. If the loss of PYY is correlatedwith development of adenocarcinoma of the colon, replacement of thepeptide has an inhibitory effect on the carcinogenic process or oncancer cells themselves. PYY expression in colon polyps and tumors hasbeen associated with increased differentiation and in fact,PYY-treatment of colon cancer cells may selectively up-regulateexpression of brush border enzymes characteristic of the normalcolonocytic phenotype. Specific, PYY-preferring Y1 receptors have alsobeen characterized in a human colon adenocarcinoma cells, suggesting thepossibility of a unique regulatory pathway for PYY. In certainembodiments, the one or more peptide YY compound(s) may be a peptide YYanalog, agonist or antagonist compound.

The few studies directly addressing the potential colon cancer growthregulatory effects of PYY have yielded varying results. For example, ina human colon adenocarcinoma cell line HT-29, PYY demonstrated no effecton cell growth or DNA synthesis. Similarly, at picomolar concentrations,PYY did not alter cell division rates or maintain hyperplastic changesin explants of azoxymethane-treated colon mucosa. In contrast, in a morerecent study, micromolar concentrations of PYY were shown to decreasecell viability in Caco-2 and HCT-116 colon cancer cells by 28 and 21%versus control, respectively. The later two studies suggest that growthregulatory effects of PYY may be dose dependent. See Tseng, et al.,Peptides 23:389-395, 2002, incorporated herein by reference.

Treatment and Prevention of Pancreatic Adenocarcinoma

As noted above, the instant invention provides improved and usefulmethods and compositions for nasal mucosal delivery of one or morepeptide YY compound(s) to prevent and treat pancreatic carcinoma andpancreatic adenocarcinoma in mammalian subjects. In a rat animal model,PYY was shown to inhibit cholecystokinin-mediated growth in the normalpancreas, as determined by pancreatic weight, RNA, and DNA content.Therefore improved and useful methods and compositions for nasal mucosaldelivery of peptide YY may exhibit growth regulatory effects inpancreatic cancer cells.

The instant invention provides improved and useful methods andcompositions for nasal mucosal delivery of one or more peptide YYcompound(s) in combination with vitamin E succinate to prevent and treatpancreatic adenocarcinoma in mammalian subjects. The role of PYY inhuman pancreatic adenocarcinoma has been examined both in vitro and invivo. Despite an early in vitro study which suggested a growthstimulatory effect, recent findings support a growth inhibitory effectin several pancreatic cancer cell lines with as little as 2.5 picomolesper liter of peptide. In addition, dramatic additive effects can occurwith simultaneous treatment using PYY and vitamin E succinate, anapoptotic inducer. Consistent with these findings, PYY has been shown toreduce pancreatic tumor size by 27% versus control in an orthotopic nudemice model. PYY-mediated growth inhibition has been postulated to occurdirectly or indirectly from reductions in intracellular cAMP levels. Incertain embodiments, the one or more peptide YY compound(s) may be apeptide YY analog, agonist or antagonist compound.

The instant invention provides improved and useful methods andcompositions for nasal mucosal delivery of synthetic analogs of peptideYY to prevent and treat pancreatic adenocarcinoma in mammalian subjects.Synthetic analogs of PYY may carry even greater potency alone and as anadjuvant to chemotherapy in pancreatic cancer. For example, BIM-43004-1is Y2 receptor-specific synthetic analog of PYY corresponding to aminoacids 22-36 with a modified N-terminus to increase cell membraneaffinity. When compared to PYY, BIM-43004-1 exhibited similar in vitrocytotoxicity in pancreatic cancer cells; however, in vivo data from nudemice demonstrated 2-fold greater decreases in tumor size, seen earlierand lasting longer at the same dosage and route of administration.Moreover, when given in combination with 5-fluorouracil and leucovorin,BIM-43004-1 reduced cell growth and expression of epidermal growthfactor receptor slightly more so than PYY. See Tseng, et al., Peptides23:389-395, 2002, incorporated herein by reference.

Treatment and Prevention of Breast Carcinoma

As noted above, the instant invention provides improved and usefulmethods and compositions for nasal mucosal delivery of one or morepeptide YY compound(s) to prevent and treat human breast carcinoma inmammalian subjects. Although normal physiologic actions of peptide YYare largely confined to the brain and gut, studies have alsodemonstrated a growth regulatory effect in human breast carcinoma. PYYtreatment reduced viability by 40% in MCF-7 breast cancer cells versuscontrol and also decreased weight and volume of subcutaneous (s.c.)tumors grown in nude mice. Substantial reduction in intracellular cAMPwere observed, implicating a causal role; however, receptors involvedare unknown.

The instant invention provides improved and useful methods andcompositions for nasal mucosal delivery of one or more peptide YYcompound(s) in combination with vitamin E succinate to prevent and treathuman breast carcinoma in mammalian subjects. Studies showed thatPYY-mediated growth inhibition is independent of estrogen receptorstatus and can be augmented, as in pancreatic cancer, by combinationtherapy with vitamin E succinate. See Tseng, et al., Peptides23:389-395, 2002, incorporated herein by reference. In certainembodiments, the one or more peptide YY compound(s) may be a peptide YYanalog, agonist or antagonist compound.

Treatment and Prevention of Malnutrition that Results from IatrogenicCauses or Cachexia Associated with Advanced Disease. Improvement inMalnutrition in Cancer Patients.

As noted above, the instant invention provides improved and usefulmethods and compositions for nasal mucosal delivery of one or morepeptide YY compound(s) to prevent and treat malnutrition in mammaliansubjects with cancer. Clinically, chemotherapy and radiation treatmentoften result in gastrointestinal toxicity, nausea, and vomiting, whichlimits appetite and predisposes patients to nutritional deficiencies.Total parenteral nutrition (TPN) is often required in patientsundergoing treatment or with advanced disease states. Unfortunately,prolonged TPN results in mucosal atrophy which increases risk ofinfection and other complications; therefore an effective stimulant ofmucosal growth is needed in many cancer patients. In certainembodiments, the one or more peptide YY compound(s) may be a peptide YYanalog, for example, peptide YY₁₋₃₆.

PYY exhibits properties which render it as a useful clinical tool in thetreatment of malnutrition problems associated with cancer. For example,several studies in dogs have demonstrated that PYY, for example, peptideYY₁₋₃₆, carries a potent pro-absorptive effect on both the small boweland colon. PYY receptors have been characterized in human adipocytes andPYY has been shown to inhibit lipolysis, an essential feature of cancercachexia. Moreover, PYY has appetite stimulating effects on the centralnervous system, which has been speculated to occur due to Y1 receptorbinding or possibly a histaminergic receptor.

In an animal model, PYY expression in non-tumor bearing rats has beenobserved to increase upon administration of cytokines or hormones whichstimulate intestinal growth. In an early study, restoration ofTPN-induced reduction in gut weight with urogastrone-EGF was selectivelyparalleled by increases in serum PYY. A subsequent study usingkeratinocyte growth factor has also demonstrated increased PYY levels inassociation with gut proliferation. Treatment of mice with IGF-I ortransgenic overexpression of growth hormone or TGF-α—all of which arestimulatory toward the gut—lead to increased PYY mRNA and peptideexpression.

To better elucidate the role of PYY in promoting normal mucosalproliferation, several studies monitored tissue changes associated withdirect exogenous administration of the peptide. An early study concludedthat PYY infused i.v. over a period of three days is not trophic to thegastrointestinal tract of rats based on wet weights and mucosalmetaphase counts. In contrast, two recent studies have reported theopposite findings. Adult mice given s.c. injections of PYY for ten daysdemonstrated increased in proximal and distal small bowel weight, DNAcontent, and protein content that were dose-dependent. Similar findingsoccurred in TPN-fed rats given i.v. PYY over a seven day course. Thedirect growth-promoting role of PYY in the gut may thus depend onfactors such as route and duration of administration. See Tseng, et al.,Peptides 23:389-395, 2002, incorporated herein by reference.

Treatment and Prevention of Cancer Cachexia

As noted above, the instant invention provides improved and usefulmethods and compositions for nasal mucosal delivery of one or morepeptide YY compound(s) to prevent and treat cancer cachexia. Up to halfof all cancer patients and over 80% of patients with gastrointestinalmalignancies will eventually develop cachexia, a debilitating conditionof involuntary tissue wasting characterized by protein loss, fatbreakdown, and anorexia. The metabolic changes seen in patients arecurrently believed to result from host and tumor produced factors, whichcreate a state similar to chronic low-grade inflammation. Cancercachexia has also been shown to be associated with decreased proteinsynthesis and protein loss in the small bowel. Although PYY may notdirectly alter the underlying cause of cachexia, PYY does increase smallbowel protein content in normal mice and rats, as mentioned. In certainembodiments, the one or more peptide YY compound(s) may be a peptide YYanalog, for example, peptide YY₁₋₃₆.

One study has examined the potential therapeutic role of PYY in thecontext of cancer cachexia. Rats bearing subcutaneous methylcholanthrenesarcomas were given intravenous. PYY, clenbuterol, an anabolic B2adrenergic agonist, or both agents together. PYY alone increased smallbowel weight and protein content, but had no effect on the colon.Combination therapy increased mean protein content in the gastrocnemiusmuscle; however these effects appear to be mediated by clenbuterolversus PYY. Most significantly no difference in tumor growth wasobserved between treatment and control groups while tumor bearing ratsgiven PYY alone exhibited higher mean body weights versus control. SeeTseng, et al., Peptides 23:389-395, 2002, incorporated herein byreference.

Treatment and Prevention of Disease and Reduction of Nasal MucosalInflammation by Intranasal Administration of Peptide YY, for Example,Human Peptide YY, in Combination with a Steroid Composition.

The treatment and prevention of disease, for example, obesity,Alzheimer's disease, colon carcinoma, colon adenocarcinoma, pancreaticcarcinoma, pancreatic adenocarcinoma, breast carcinoma, treatment andprevention of malnutrition resulting from iatrogenic causes or cachexiaassociated with advanced disease, or cancer cachexia by therapy withintranasal compositions of peptide YY and corticosteroid, as describedherein, results in reduction in disease indications while avoiding sideeffects of drug delivery. Intranasal compositions of peptide YY andcorticosteroid results in reduced nasal irritation, reduced rhinitis anda reduced nasal mucosal allergic response by direct delivery to thenasal mucosal tissue and to the CNS tissue or fluid. Direct intranasaldelivery of the compositions to the CNS tissue or fluid avoids deliveryto sites of the body other than the CNS and avoids systemic sideeffects, such as adrenosuppression and weight gain, associated withsystemic delivery of corticosteroids to the blood serum and organs, forexample, the adrenal gland and kidney.

Mucosal administration of the peptide YY and corticosteroid compositionsonce or twice per day for 7 to 14 days to the subject yields extendeddelivery of the peptide YY and corticosteroid compositions. Delivery ofthe composition is measured by area under the concentration curve (AUC)for peptide YY, the corticosteroid, or for a pharmacokinetic marker forpeptide YY. Mucosal administration of the peptide YY and steroidcompositions to the subject yields an AUC of corticosteroid or peptideYY in a central nervous system (CNS) tissue or fluid of the subject thatis typically about 50%, about 75% or about 100% or greater compared toan AUC of corticosteroid or peptide YY in CNS tissue or fluid followingsubcutaneous injection of an equivalent concentration or dose of peptideYY to the subject.

A pharmaceutical formulation suitable for intranasal administrationcomprising peptide YY and a corticosteroid compound for treatment ofinflammation, as described herein, provides therapeutic delivery to theCNS while avoiding delivery to the blood serum and organs, for example,adrenal gland and kidneys. Pharmaceutical compositions yield an areaunder the concentration curve (AUC) of a corticosteroid composition inthe CNS that is typically about 2-fold, about 3-fold, about 5-fold, orabout 10-fold or greater when compared to an AUC for the composition ina blood plasma or other target tissue (adrenal gland or kidney).Pharmaceutical formulations, as described herein, target corticosteroidsto the CNS tissues and fluids thus avoiding adverse steroid sideeffects, such as adrenosuppression and weight gain caused by prolongedsteroid treatment.

Methods and Compositions of Delivery

Improved methods and compositions for mucosal administration of peptideYY to mammalian subjects optimize peptide YY dosing schedules. Thepresent invention provides mucosal delivery of peptide YY formulatedwith one or more mucosal delivery-enhancing agents wherein peptide YYdosage release is substantially normalized and/or sustained for aneffective delivery period of peptide YY release ranges fromapproximately 0.1 to 2.0 hours; 0.4 to 1.5 hours; 0.7 to 1.5 hours; or0.8 to 1.0 hours; following mucosal administration. The sustainedrelease of peptide YY is achieved may be facilitated by repeatedadministration of exogenous peptide YY utilizing methods andcompositions of the present invention.

Compositions and Methods of Sustained Release

Improved compositions and methods for mucosal administration of peptideYY to mammalian subjects optimize peptide YY dosing schedules. Thepresent invention provides improved mucosal (e.g., nasal) delivery of aformulation comprising peptide YY in combination with one or moremucosal delivery-enhancing agents and an optional sustainedrelease-enhancing agent or agents. Mucosal delivery-enhancing agents ofthe present invention yield an effective increase in delivery, e.g., anincrease in the maximal plasma concentration (C_(max)) to enhance thetherapeutic activity of mucosally-administered peptide YY. A secondfactor affecting therapeutic activity of peptide YY in the blood plasmaand CNS is residence time (RT). Sustained release-enhancing agents, incombination with intranasal delivery-enhancing agents, increase C_(max)and increase residence time (RT) of peptide YY. Polymeric deliveryvehicles and other agents and methods of the present invention thatyield sustained release-enhancing formulations, for example,polyethylene glycol (PEG), are disclosed herein. The present inventionprovides an improved peptide YY delivery method and dosage form fortreatment of symptoms related to obesity, colon cancer, pancreaticcancer, or breast cancer in mammalian subjects.

Maintenance of Basal Levels of Peptide YY

Improved compositions and methods for mucosal administration of peptideYY to mammalian subjects optimize peptide YY dosing schedules. Thepresent invention provides improved nasal mucosal delivery of aformulation comprising peptide YY and intranasal delivery-enhancingagents in combination with intramuscular or subcutaneous administrationof peptide YY. Formulations and methods of the present inventionmaintain relatively consistent basal levels of peptide YY, for example,at a basal level of 15-80 pM when administered by nasal mucosal deliveryof a formulation comprising peptide YY and intranasal delivery-enhancingagents of the present invention, alone, or in combination withintramuscular or subcutaneous administration of peptide YY. Basal levelsof peptide YY are maintained throughout a 2 to 24 hour, 4-16 hour, or8-12 hour period following a single dose administration or attended by amultiple dosing regimen of 2-6 sequential administrations. Maintenanceof basal levels of peptide YY is particularly useful for treatment andprevention of disease, for example, multiple sclerosis, withoutunacceptable adverse side effects.

Within the mucosal delivery formulations and methods of the invention,the peptide YY is frequently combined or coordinately administered witha suitable carrier or vehicle for mucosal delivery. As used herein, theterm “carrier” means a pharmaceutically acceptable solid or liquidfiller, diluent or encapsulating material. A water-containing liquidcarrier can contain pharmaceutically acceptable additives such asacidifying agents, alkalizing agents, antimicrobial preservatives,antioxidants, buffering agents, chelating agents, complexing agents,solubilizing agents, humectants, solvents, suspending and/orviscosity-increasing agents, tonicity agents, wetting agents or otherbiocompatible materials. A tabulation of ingredients listed by the abovecategories, can be found in the U.S. Pharmacopeia National Formulary,1857-1859, 1990, which is incorporated herein by reference. Someexamples of the materials which can serve as pharmaceutically acceptablecarriers are sugars, such as lactose, glucose and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols such as glycerin, sorbitol, mannitoland polyethylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer'ssolution, ethyl alcohol and phosphate buffer solutions, as well as othernon toxic compatible substances used in pharmaceutical formulations.Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfateand magnesium stearate, as well as coloring agents, release agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions,according to the desires of the formulator. Examples of pharmaceuticallyacceptable antioxidants include water soluble antioxidants such asascorbic acid, cysteine hydrochloride, sodium bisulfite, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidantssuch as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and thelike; and metal-chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid andthe like. The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form will vary dependingupon the particular mode of administration.

The mucosal formulations of the invention are generally sterile,particulate free and stable for pharmaceutical use. As used herein, theterm “particulate free” means a formulation that meets the requirementsof the USP specification for small volume parenteral solutions. The term“stable” means a formulation that fulfills all chemical and physicalspecifications with respect to identity, strength, quality, and puritywhich have been established according to the principles of GoodManufacturing Practice, as set forth by appropriate governmentalregulatory bodies.

Within the mucosal delivery compositions and methods of the invention,various delivery-enhancing agents are employed which enhance delivery ofpeptide YY into or across a mucosal surface. In this regard, delivery ofpeptide YY across the mucosal epithelium can occur “transcellularly” or“paracellularly.” The extent to which these pathways contribute to theoverall flux and bioavailability of the peptide YY depends upon theenvironment of the mucosa, the physico-chemical properties the activeagent, and on the properties of the mucosal epithelium. Paracellulartransport involves only passive diffusion, whereas transcellulartransport can occur by passive, facilitated or active processes.Generally, hydrophilic, passively transported, polar solutes diffusethrough the paracellular route, while more lipophilic solutes use thetranscellular route. Absorption and bioavailability (e.g., as reflectedby a permeability coefficient or physiological assay), for diverse,passively and actively absorbed solutes, can be readily evaluated, interms of both paracellular and transcellular delivery components, forany selected peptide YY within the invention. These values can bedetermined and distinguished according to well known methods, such as invitro epithelial cell culture permeability assays. Hilgers, et al.,Pharm. Res. 7:902-910, 1990; Wilson, et al., J. Controlled Release11:25-40, 1990; Artursson, I., Pharm. Sci. 79:476-482, 1990; Cogburn, etal., Pharm. Res. 8:210-216, 1991; Pade, et al., Pharmaceutical Research14:1210-1215, 1997, each incorporated herein by reference.

For passively absorbed drugs, the relative contribution of paracellularand transcellular pathways to drug transport depends upon the pKa,partition coefficient, molecular radius and charge of the drug, the pHof the luminal environment in which the drug is delivered, and the areaof the absorbing surface. The paracellular route represents a relativelysmall fraction of accessible surface area of the nasal mucosalepithelium. In general terms, it has been reported that cell membranesoccupy a mucosal surface area that is a thousand times greater than thearea occupied by the paracellular spaces. Thus, the smaller accessiblearea, and the size- and charge-based discrimination againstmacromolecular permeation would suggest that the paracellular routewould be a generally less favorable route than transcellular deliveryfor drug transport. Surprisingly, the methods and compositions of theinvention provide for significantly enhanced transport ofbiotherapeutics into and across mucosal epithelia via the paracellularroute. Therefore, the methods and compositions of the inventionsuccessfully target both paracellular and transcellular routes,alternatively or within a single method or composition.

As used herein, “mucosal delivery-enhancing agents” include agents whichenhance the release or solubility (e.g., from a formulation deliveryvehicle), diffusion rate, penetration capacity and timing, uptake,residence time, stability, effective half-life, peak or sustainedconcentration levels, clearance and other desired mucosal deliverycharacteristics (e.g., as measured at the site of delivery, or at aselected target site of activity such as the bloodstream or centralnervous system) of peptide YY or other biologically active compound(s).Enhancement of mucosal delivery can thus occur by any of a variety ofmechanisms, for example by increasing the diffusion, transport,persistence or stability of peptide YY, increasing membrane fluidity,modulating the availability or action of calcium and other ions thatregulate intracellular or paracellular permeation, solubilizing mucosalmembrane components (e.g., lipids), changing non-protein and proteinsulfhydryl levels in mucosal tissues, increasing water flux across themucosal surface, modulating epithelial junctional physiology, reducingthe viscosity of mucus overlying the mucosal epithelium, reducingmucociliary clearance rates, and other mechanisms.

As used herein, a “mucosally effective amount of peptide YY”contemplates effective mucosal delivery of peptide YY to a target sitefor drug activity in the subject that may involve a variety of deliveryor transfer routes. For example, a given active agent may find its waythrough clearances between cells of the mucosa and reach an adjacentvascular wall, while by another route the agent may, either passively oractively, be taken up into mucosal cells to act within the cells or bedischarged or transported out of the cells to reach a secondary targetsite, such as the systemic circulation. The methods and compositions ofthe invention may promote the translocation of active agents along oneor more such alternate routes, or may act directly on the mucosal tissueor proximal vascular tissue to promote absorption or penetration of theactive agent(s). The promotion of absorption or penetration in thiscontext is not limited to these mechanisms.

As used herein “peak concentration (C_(max)) of peptide YY in a bloodplasma”, “area under concentration vs. time curve (AUC) of peptide YY ina blood plasma”, “time to maximal plasma concentration (t_(max)) ofpeptide YY in a blood plasma” are pharmacokinetic parameters known toone skilled in the art. Laursen, et al., Eur. J. Endocrinology135:309-315, 1996, incorporated herein by reference. The “concentrationvs. time curve” measures the concentration of peptide YY in a bloodserum of a subject vs. time after administration of a dosage of peptideYY to the subject either by intranasal, intramuscular, subcutaneous, orother parenteral route of administration. “C_(max)” is the maximumconcentration of peptide YY in the blood serum of a subject following asingle dosage of peptide YY to the subject. “t_(max)” is the time toreach maximum concentration of peptide YY in a blood serum of a subjectfollowing administration of a single dosage of peptide YY to thesubject.

As used herein, “area under concentration vs. time curve (AUC) ofpeptide YY in a blood plasma” is calculated according to the lineartrapezoidal rule and with addition of the residual areas. A decrease of23% or an increase of 30% between two dosages would be detected with aprobability of 90% (type II error β=10%). The “delivery rate” or “rateof absorption” is estimated by comparison of the time (t_(max)) to reachthe maximum concentration (C_(max)). Both C_(max) and t_(max) areanalyzed using non-parametric methods. Comparisons of thepharmacokinetics of intramuscular, subcutaneous, intravenous andintranasal peptide YY administrations were performed by analysis ofvariance (ANOVA). For pairwise comparisons a Bonferroni-Holmessequential procedure was used to evaluate significance. Thedose-response relationship between the three nasal doses was estimatedby regression analysis. P<0.05 was considered significant. Results aregiven as mean values +/−SEM. (Laursen, et al., 1996.)

Pharmacokinetic parameters and values described herein, includingt_(max), C_(max), and AUC may be determined by comparing an intranasaldelivery formulation or method of the invention to apreviously-described “control” delivery method, for example apreviously-described mucosal delivery, intramuscular delivery,subcutaneous delivery, intravenous delivery, and/or parenteral deliverymethod different formulations or delivery methods involving a commonroute of delivery. In alternate embodiments, pharmacokinetic and/orpharmacodynamic characteristics or values may be expressed, distinctlyfrom other embodiments, by comparing an intranasal method or formulationof the invention with a different intranasal delivery method orformulation. For example, a method or formulation may be compared with apreviously-described “control” method or formulation involving adifferent administration protocol, or formulation component. Wheredifferent formulations, administration protocols, and/or differentpharmacokinetic or pharmacodynamic controls are used herein to determinea comparative pharmacokinetic value or range for one embodiment oraspect of the invention, such values or ranges are not intended to beinterchangeable or directly correlated between different embodiments andaspects of the invention. Rather, various embodiments and aspects of theinvention are disclosed herein that each may have be characterized bydistinct pharmacokinetic and or pharmacodynamic characteristics and/orvalues, whereby the characteristics and values may not be consistent orcomparable among the different embodiments or aspects.

As used herein, “pharmacokinetic markers” include any acceptedbiological marker that is detectable in an in vitro or in vivo systemuseful for modeling pharmacokinetics of mucosal delivery of one or morepeptide YY compounds, or other biologically active agent(s) disclosedherein, wherein levels of the marker(s) detected at a desired targetsite following administration of the peptide YY compound(s) according tothe methods and formulations herein, provide a reasonably correlativeestimate of the level(s) of the peptide YY compound(s) delivered to thetarget site. Among many art-accepted markers in this context aresubstances induced at the target site by administration of the peptideYY compound(s) or other biologically active agent(s). For example, nasalmucosal delivery of an effective amount of one or more peptide YYcompounds according to the invention stimulates an immunologic responsein the subject measurable by production of pharmacokinetic markers.

Many known reagents that are reported to enhance mucosal absorption alsocause irritation or damage to mucosal tissues. Swenson and Curatolo,Adv. Drug Delivery Rev. 8:39-92, 1992, incorporated herein by reference.For example, in studies of intestinal absorption enhancing agents, thedelivery-enhancing effects of various absorption-promoting agents arereportedly directly related to their membrane toxicity. Uchiyama, etal., Biol. Pharm. Bull. 19:1618-1621, 1996; Yamamoto, et al., J. Pharm.Pharmacol. 48:1285-1289, 1996, each incorporated herein by reference. Inthis regard, the combinatorial formulation and coordinate administrationmethods of the present invention incorporate effective, minimally toxicdelivery-enhancing agents to enhance mucosal delivery of peptide YY andother biologically active macromolecules useful within the invention.

While the mechanism of absorption promotion may vary with differentintranasal delivery-enhancing agents of the invention, useful reagentsin this context will not substantially adversely affect the mucosaltissue and will be selected according to the physicochemicalcharacteristics of the particular peptide YY or other active ordelivery-enhancing agent. In this context, delivery enhancing agentsthat increase penetration or permeability of mucosal tissues will oftenresult in some alteration of the protective permeability barrier of themucosa. For such delivery-enhancing agents to be of value within theinvention, it is generally desired that any significant changes inpermeability of the mucosa be reversible within a time frame appropriateto the desired duration of drug delivery. Furthermore, there should beno substantial, cumulative toxicity, nor any permanent deleteriouschanges induced in the barrier properties of the mucosa with long-termuse.

Within certain aspects of the invention, absorption-promoting agents forcoordinate administration or combinatorial formulation with peptide YYof the invention are selected from small hydrophilic molecules,including but not limited to, dimethyl sulfoxide (DMSO),dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones.Alternatively, long-chain amphipathic molecules, for example,deacylmethyl sulfoxide, azone, sodium laurylsulfate, oleic acid, and thebile salts, may be employed to enhance mucosal penetration of thepeptide YY. In additional aspects, surfactants (e.g., polysorbates) areemployed as adjunct compounds, processing agents, or formulationadditives to enhance intranasal delivery of the peptide YY. Thesepenetration enhancing agents typically interact at either the polar headgroups or the hydrophilic tail regions of molecules which comprise thelipid bilayer of epithelial cells lining the nasal mucosa. Barry,Pharmacology of the Skin 1: 121-137; Shroot, et al., eds., Karger,Basel, 1987; and Barry, J. Controlled Release 6:85-97, 1987, eachincorporated herein by reference. Interaction at these sites may havethe effect of disrupting the packing of the lipid molecules, increasingthe fluidity of the bilayer, and facilitating transport of the peptideYY across the mucosal barrier. Interaction of these penetrationenhancers with the polar head groups may also cause or permit thehydrophilic regions of adjacent bilayers to take up more water and moveapart, thus opening the paracellular pathway to transport of the peptideYY. In addition to these effects, certain enhancers may have directeffects on the bulk properties of the aqueous regions of the nasalmucosa. Agents such as DMSO, polyethylene glycol, and ethanol can, ifpresent in sufficiently high concentrations in delivery environment(e.g., by pre-administration or incorporation in a therapeuticformulation), enter the aqueous phase of the mucosa and alter itssolubilizing properties, thereby enhancing the partitioning of thepeptide YY from the vehicle into the mucosa.

Additional mucosal delivery-enhancing agents that are useful within thecoordinate administration and processing methods and combinatorialformulations of the invention include, but are not limited to, mixedmicelles; enamines; nitric oxide donors (e.g.,S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4-which are preferablyco-administered with an NO scavenger such as carboxy-PITO or doclofenacsodium); sodium salicylate; glycerol esters of acetoacetic acid (e.g.,glyceryl-1,3-diacetoacetate or1,2-isopropylideneglycerine-3-acetoacetate); and other release-diffusionor intra- or trans-epithelial penetration-promoting agents that arephysiologically compatible for mucosal delivery. Otherabsorption-promoting agents are selected from a variety of carriers,bases and excipients that enhance mucosal delivery, stability, activityor trans-epithelial penetration of the peptide YY. These include, interalia, cyclodextrins and β-cyclodextrin derivatives (e.g.,2-hydroxypropyl-β-cyclodextrin andheptakis(2,6-di-O-methyl-β-cyclodextrin). These compounds, optionallyconjugated with one or more of the active ingredients and furtheroptionally formulated in an oleaginous base, enhance bioavailability inthe mucosal formulations of the invention. Yet additionalabsorption-enhancing agents adapted for mucosal delivery includemedium-chain fatty acids, including mono- and diglycerides (e.g., sodiumcaprate—extracts of coconut oil, Capmul), and triglycerides (e.g.,amylodextrin, Estaram 299, Miglyol 810).

The mucosal therapeutic and prophylactic compositions of the presentinvention may be supplemented with any suitable penetration-promotingagent that facilitates absorption, diffusion, or penetration of peptideYY across mucosal barriers. The penetration promoter may be any promoterthat is pharmaceutically acceptable. Thus, in more detailed aspects ofthe invention compositions are provided that incorporate one or morepenetration-promoting agents selected from sodium salicylate andsalicylic acid derivatives (acetyl salicylate, choline salicylate,salicylamide, etc.); amino acids and salts thereof (e.g.,monoaminocarboxlic acids such as glycine, alanine, phenylalanine,proline, hydroxyproline, etc.; hydroxyamino acids such as serine; acidicamino acids such as aspartic acid, glutamic acid, etc.; and basic aminoacids such as lysine, etc. —inclusive of their alkali metal or alkalineearth metal salts); and N-acetylamino acids (N-acetylalanine,N-acetylphenylalanine, N-acetylserine, N-acetylglycine, N-acetyllysine,N-acetylglutamic acid, N-acetylproline, N-acetylhydroxyproline, etc.)and their salts (alkali metal salts and alkaline earth metal salts).Also provided as penetration-promoting agents within the methods andcompositions of the invention are substances which are generally used asemulsifiers (e.g., sodium oleyl phosphate, sodium lauryl phosphate,sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene alkylethers, polyoxyethylene alkyl esters, etc.), caproic acid, lactic acid,malic acid and citric acid and alkali metal salts thereof,pyrrolidonecarboxylic acids, alkylpyrrolidonecarboxylic acid esters,N-alkylpyrrolidones, proline acyl esters, and the like.

Within various aspects of the invention, improved nasal mucosal deliveryformulations and methods are provided that allow delivery of peptide YYand other therapeutic agents within the invention across mucosalbarriers between administration and selected target sites. Certainformulations are specifically adapted for a selected target cell, tissueor organ, or even a particular disease state. In other aspects,formulations and methods provide for efficient, selective endo- ortranscytosis of peptide YY specifically routed along a definedintracellular or intercellular pathway. Typically, the peptide YY isefficiently loaded at effective concentration levels in a carrier orother delivery vehicle, and is delivered and maintained in a stabilizedform, e.g., at the nasal mucosa and/or during passage throughintracellular compartments and membranes to a remote target site fordrug action (e.g., the blood stream or a defined tissue, organ, orextracellular compartment). The peptide YY may be provided in a deliveryvehicle or otherwise modified (e.g., in the form of a prodrug), whereinrelease or activation of the peptide YY is triggered by a physiologicalstimulus (e.g., pH change, lysosomal enzymes, etc.) Often, the peptideYY is pharmacologically inactive until it reaches its target site foractivity. In most cases, the peptide YY and other formulation componentsare non-toxic and non-immunogenic. In this context, carriers and otherformulation components are generally selected for their ability to berapidly degraded and excreted under physiological conditions. At thesame time, formulations are chemically and physically stable in dosageform for effective storage.

Biologically Active Agents

The methods and compositions of the present invention are directedtoward enhancing mucosal, e.g., intranasal, delivery of peptide YY orother biologically active agents to achieve therapeutic, prophylactic orother desired physiological results in mammalian subjects. As usedherein, the term “biologically active agent” encompasses any substancethat produces a physiological response when mucosally administered to amammalian subject according to the methods and compositions herein.Useful biologically active agents in this context include therapeutic orprophylactic agents applied in all major fields of clinical medicine, aswell as nutrients, cofactors, enzymes (endogenous or foreign),antioxidants, and the like. Thus, the biologically active agent may bewater-soluble or water-insoluble, and may include higher molecularweight proteins, peptides, carbohydrates, glycoproteins, lipids, and/orglycolipids, nucleosides, polynucleotides, and other active agents.

Peptide and Protein Analogs and Mimetics

Included within the definition of biologically active peptides andproteins for use within the invention are natural or synthetic,therapeutically or prophylactically active, peptides (comprised of twoor more covalently linked amino acids), proteins, peptide or proteinfragments, peptide or protein analogs, and chemically modifiedderivatives or salts of active peptides or proteins. A wide variety ofuseful analogs and mimetics of peptide YY are contemplated for usewithin the invention and can be produced and tested for biologicalactivity according to know methods. Often, the peptides or proteins ofpeptide YY or other biologically active peptides or proteins for usewithin the invention are muteins that are readily obtainable by partialsubstitution, addition, or deletion of amino acids within a naturallyoccurring or native (e.g., wild-type, naturally occurring mutant, orallelic variant) peptide or protein sequence. Additionally, biologicallyactive fragments of native peptides or proteins are included. Suchmutant derivatives and fragments substantially retain the desiredbiological activity of the native peptide or proteins. In the case ofpeptides or proteins having carbohydrate chains, biologically activevariants marked by alterations in these carbohydrate species are alsoincluded within the invention.

“Peptide YY” or “PYY” refers to “peptide YY” in native-sequence or invariant form, and from any source, whether natural, synthetic, orrecombinant. Peptide YY is a human peptide YY, for example, Peptide YY(human: (SEQ ID NO: 792)H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂);(Leu³¹,Pro³⁴)-Peptide YY (human: (SEQ ID NO: 798)H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Pro-Arg-Tyr-NH₂);(Pro³⁴)-Peptide YY (human: (SEQ ID NO: 799)H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Pro-Arg-Tyr-NH₂);Peptide YY₃₋₃₆ (human: (SEQ ID NO: 794)H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂);Peptide YY₁₃₋₃₆ (porcine, rat: (SEQ ID NO: 793)H-Ser-Pro-Glu-Glu-Leu-Ser-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂);(Bachem AG, King of Prussia, Pa., United States).

Examples include human peptide YY which is natural or recombinant.Recombinant peptide YY is synthesized in an E. coli bacterial proteinsynthesis process, or in a protein synthesis process in yeast cells,baculovirus in insect cells, or mammalian cells, for example, CHO or BHKcells.

“Pancreatic polypeptide (PP)” or “neuropeptide Y (NPY)” has peptideYY-like activity, or may be a peptide YY agonist binding to a “Yreceptor.” In certain formulations, “pancreatic polypeptide (PP)” or“neuropeptide Y (NPY)” may be substituted for peptide YY.

Biologically active peptides and proteins for use within the methods andcompositions of the invention thus include native or “wild-type”peptides and proteins and naturally occurring variants of thesemolecules, e.g., naturally occurring allelic variants and mutantproteins. Also included are synthetic, e.g., chemically or recombinantlyengineered, peptides and proteins, as well as peptide and protein“analogs” and chemically modified derivatives, fragments, conjugates,and polymers of naturally occurring peptides and proteins. As usedherein, the term peptide or protein “analog” is meant to includemodified peptides and proteins incorporating one or more amino acidsubstitutions, insertions, rearrangements or deletions as compared to anative amino acid sequence of a selected peptide or protein, or of abinding domain, fragment, immunogenic epitope, or structural motif, of aselected peptide or protein. Peptide and protein analogs thus modifiedexhibit substantially conserved biological activity comparable to thatof a corresponding native peptide or protein, which means activity(e.g., specific binding to a peptide YY protein, or to a cell expressingsuch a protein, specific ligand or receptor binding activity, etc.)levels of at least 50%, typically at least 75%, often 85%-95% orgreater, compared to activity levels of a corresponding native proteinor peptide.

For purposes of the present invention, the term biologically activepeptide or protein “analog” further includes derivatives or syntheticvariants of a native peptide or protein, such as amino and/or carboxylterminal deletions and fusions, as well as intrasequence insertions,substitutions or deletions of single or multiple amino acids.Insertional amino acid sequence variants are those in which one or moreamino acid residues are introduced into a predetermined site in theprotein. Random insertion is also possible with suitable screening ofthe resulting product. Deletional variants are characterized by removalof one or more amino acids from the sequence. Substitutional amino acidvariants are those in which at least one residue in the sequence hasbeen removed and a different residue inserted in its place.

Where a native peptide or protein is modified by amino acidsubstitution, amino acids are generally replaced by other amino acidshaving similar, conservatively related chemical properties such ashydrophobicity, hydrophilicity, electronegativity, small or bulky sidechains, and the like. Residue positions which are not identical to thenative peptide or protein sequence are thus replaced by amino acidshaving similar chemical properties, such as charge or polarity, wheresuch changes are not likely to substantially effect the properties ofthe peptide or protein analog. These and other minor alterations willtypically substantially maintain biological properties of the modifiedpeptide or protein, including biological activity (e.g., binding topeptide YY, adhesion molecule, or other ligand or receptor),immunoidentity (e.g., recognition by one or more monoclonal antibodiesthat recognize a native peptide or protein), and other biologicalproperties of the corresponding native peptide or protein.

As used herein, the term “conservative amino acid substitution” refersto the general interchangeability of amino acid residues having similarside chains. For example, a commonly interchangeable group of aminoacids having aliphatic side chains is alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Examples of conservativesubstitutions include the substitution of a non-polar (hydrophobic)residue such as isoleucine, valine, leucine or methionine for another.Likewise, the present invention contemplates the substitution of a polar(hydrophilic) residue such as between arginine and lysine, betweenglutamine and asparagine, and between threonine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another or the substitution of an acidicresidue such as aspartic acid or glutamic acid for another is alsocontemplated. Exemplary conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

By aligning a peptide or protein analog optimally with a correspondingnative peptide or protein, and by using appropriate assays, e.g.,adhesion protein or receptor binding assays, to determine a selectedbiological activity, one can readily identify operable peptide andprotein analogs for use within the methods and compositions of theinvention. Operable peptide and protein analogs are typicallyspecifically immunoreactive with antibodies raised to the correspondingnative peptide or protein. Likewise, nucleic acids encoding operablepeptide and protein analogs will share substantial sequence identity asdescribed above to a nucleic acid encoding the corresponding nativepeptide or protein, and will typically selectively hybridize to apartial or complete nucleic acid sequence encoding the correspondingnative peptide or protein, or fragment thereof, under accepted, moderateor high stringency hybridization conditions. Sambrook, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratories, ColdSpring Harbor, N.Y., 1989, incorporated herein by reference. The phrase“selectively hybridizing to” refers to a selective interaction between anucleic acid probe that hybridizes, duplexes or binds preferentially toa particular target DNA or RNA sequence, for example when the targetsequence is present in a heterogenous preparation such as total cellularDNA or RNA. Generally, nucleic acid sequences encoding biologicallyactive peptide and protein analogs, or fragments thereof, will hybridizeto nucleic acid sequences encoding the corresponding native peptide orprotein under stringent conditions (e.g., selected to be about 5° C.lower than the thermal melting point (Tm) for the subject sequence at adefined ionic strength and pH, where the Tm is the temperature underdefined ionic strength and pH at which 50% of the complementary ortarget sequence hybridizes to a perfectly matched probe). Fordiscussions of nucleic acid probe design and annealing conditions, see,for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd ed., Vols. 1-3, Cold Spring Harbor Laboratory, 1989 or CurrentProtocols in Molecular Biology, Ausubel, F., et al., ed., GreenePublishing and Wiley-Interscience, New York, 1987, each of which isincorporated herein by reference. Typically, stringent or selectiveconditions will be those in which the salt concentration is at leastabout 0.02 molar at pH 7 and the temperature is at least about 60° C.Less stringent selective hybridization conditions may also be chosen. Asother factors may significantly affect the stringency of hybridization,including, among others, base composition and size of the complementarystrands, the presence of organic solvents and the extent of basemismatching, the combination of parameters is more important than thespecific measure of any one.

Aggregation Inhibitory Agents and Methods

Protein aggregation is of major importance in biotechnology for the invitro production and in vivo use of recombinant peptides and proteins,including peptide YY and other active peptides and proteins for usewithin the invention. Aggregation commonly limits the stability,solubility and yields of recombinant proteins for use in pharmaceuticalformulations. Under various conditions, therapeutic peptides andproteins for use within the invention may exhibit functionallydeleterious aggregation. Commonly, peptides and proteins expressed inlarge quantities in heterologous expression systems precipitate withinthe recombinant host cell in dense aggregates. Such insoluble aggregatesof expressed polypeptide (inclusion bodies) may reflect improperlyfolded polypeptides relating to the inability of the host cell toproperly process and/or secrete the recombinant polypeptide. Theaggregated fraction often constitutes a major fraction of total cellprotein in recombinant expression systems. Further details of peptideand protein aggregation are provided in Brems, et al., Biochemistry24:7662, 1985; Mitraki, et al., Bio/Technology 7:690, 1989; Marston andHartley, Meth. Enzymol. 182:264-276, 1990; Wetzel, “Protein AggregationIn vivo: Bacterial Inclusion Bodies and Mammalian Amyloid,” in Stabilityof Protein Pharmaceuticals: In vivo Pathways of Degradation andStrategies for Protein Stabilization, Ahern and Manning (eds.) (PlenumPress, 1991); and Wetzel, “Enhanced Folding and Stabilization ofProteins by Suppression of Aggregation In vitro and In vivo,” in ProteinEngineering—A Practical Approach, Rees, A. R., et al. (eds.) (IRL Pressat Oxford University Press, Oxford, 1991) each of the foregoingpublications is incorporated herein by reference.

Recovery of therapeutic peptides and proteins from aggregate forms,e.g., as found in recombinant expression systems, presents numerousproblems. In many cases, peptides and proteins recovered from aggregatesare predominantly biologically inactive, often because they folded intoa three-dimensional conformation different from that of native protein.Misfolding can occur either in the cell during fermentation or duringprotein isolation, processing or storage procedures. Methods forpreventing aggregation, and for isolating and refolding proteins fromaggregated complexes into a correct, biologically active conformation,are therefore important for obtaining functional peptides and proteinsfor therapeutic use within the invention.

Accordingly, the present invention provides compositions and methodsthat are effective in producing or maintaining “unaggregated” peptidesor proteins in a pharmaceutical formulation for mucosal delivery of thesubject peptide(s) or protein(s). The methods involve solubilizingpeptides and proteins from aggregates and/or stabilizing peptides andproteins that are prone to aggregation—to provide formulations ofsoluble, stable, biologically active peptide or protein suitable formucosal, e.g., intranasal, administration. Such formulations contain asolubilized peptide or protein in a substantially pure, unaggregated andtherapeutically useful form.

Typically, the peptide or protein which is solubilized from aggregate orstabilized to reduce aggregation is initially obtained from arecombinant expression system, often from insoluble aggregate form. Thelatter procedure typically involves disruption of the host cells andseparation of the ruptured cell materials from the insolubilized protein(as inclusion bodies). Examples of available means for accomplishingthis are procedures involving the use of sonication and homogenizationin the presence of one or more detergents and separation of the rupturedcell materials from the aggregated peptide or protein by centrifugation(see, e.g., U.S. Pat. Nos. 4,828,929 and 4,673,641). It should beunderstood that other well known procedures can be also be used in thiscontext.

Peptides or proteins recovered from recombinant systems in this mannertypically comprise a broad spectrum of polypeptides ranging from solublemonomers and multimers to macroscopic insoluble structures in whichthousands of such individual polypeptide fragments are bound. Typically,however, those aggregates composed of approximately 10 to 20, or fewerfragments, and having a molecular weight of 200,000 to 400,000 aresoluble. Such fragments, which are referred to herein as “solubleaggregate,” have relatively low therapeutic utility as measured in invitro assays. Certain even larger complexes are also soluble, althoughalso of relatively low therapeutic utility.

As used herein, “unaggregated” peptide or protein comprises a peptide orprotein composition that is substantially free of aggregate, whethersoluble or insoluble. The composition of unaggregated peptide or proteintypically comprises a population of monomeric peptide or protein, butmay also include noncovalently linked multimeric species. Typically, theamount of “soluble aggregate” present in such samples (e.g., asdetermined by high performance liquid chromatography (HPLC)) is lessthan about 15%, often less than about 5%, and commonly less than about0.5% of the subject peptide or protein species in a preparation. Inalternate terms, the compositions of the invention are “substantiallyfree of aggregate,” wherein the percent by weight of monomer in apurified peptide or protein preparation is at least about 40% to 65%,more typically about 65% to 80 weight %, often at least 75%-95% orgreater.

For some peptides and proteins, the formation of inclusion bodies andother types of insoluble aggregates may be related to the presence ofcysteine residues in the subject peptide or protein. It is believed thatincorrect disulfide bonds are encouraged to form either within inclusionbodies or during attempts to solubilize the polypeptides there from, aswell as under other purification or storage conditions. When such bondsare formed within a polypeptide (an intrachain bond), they may lead to abiologically inactive conformation of the molecule. When disulfide bondsare formed between fragments (an interchain bond), they may lead toinsoluble or biologically inactive dimers or aggregates. Illustrative ofthis phenomenon, misfolded IGF-I possesses different disulfide bondpairs than are found in native IGF-I, and exhibits significantly reducedbiological activity (Raschdorf, et al., Biomedical and EnvironmentalMass Spectroscopy 16:3-8, 1988, incorporated herein by reference). Inother cases, proteins isolated from aggregates produce disulfide-linkeddimers, trimers, and multimers. Morris, et al., Biochem. J. 268:803-806,1990; Toren, et al., Anal. Biochem. 169:287-299, 1988; Frank, et al., inPeptides: synthesis-structure-function, ed. Rich, D. H. and E. Gross,pp. 729-738 (Pierce Chemical Company, Rockford, Ill., 1981), eachincorporated herein by reference. This association phenomenon is verycommon during protein refolding, particularly at higher proteinconcentrations, and appears to often involve association throughhydrophobic interaction of partially folded intermediates. Cleland andWang, Biochemistry 29:11072-11078, 1990, incorporated herein byreference.

Thus, successful manipulation of mammalian proteins expressed fromrecombinant bacterial systems has generally required that the cysteineresidues thereof be altered so that they cannot react with othercysteine residues. Without this treatment, undesired reaction of thecysteine residues thereof typically occurs, leading to the formation ofinsoluble or biologically inactive polypeptide aggregates unsuited foreffective use as therapeutics.

There are numerous well-known procedures that can be used within theinvention to successfully alter cysteine residues of therapeuticpeptides and proteins that are prone to aggregation involving disulfidebonding. One such technique involves treatment of cysteine residues witha reducing agent such as; for example, beta-mercaptoethanol ordithiothreitol (DTT) followed by permanent alkylation (for example, withiodoacetamide) of the cysteine residues. Numerous other covalent labelsmay be attached to the target cysteine residues, so long as they areapplied under pH conditions that do not irreversibly denature the targetpeptide or protein and do not allow chemical reaction with othercysteine residues. Such covalent labeling procedures are generally knownin the art and include also, for example, reaction with iodoacetic acidor iodinating agents such as iodofluorescein. Additionally, cysteineresidues may be chemically altered such as by sulfitolyzation.Alteration can be accomplished also by site directed mutagenesis of anencoding DNA, replacing cysteine residues with “inert” residues such as,for example, glycine or alanine, or by deletion of sequence positionscorresponding to cysteine. A sufficient number of the cysteine residuesare altered to avoid the aggregation problems caused by their presence.For additional details regarding methods for preparing cysteine-alteredproteins to minimize aggregation, see, e.g., U.S. Pat. No. 5,847,086,incorporated herein by reference.

For methods that do not involve cysteine modification, it is importantto note that protein folding is influenced by the nature of the mediumcontaining the protein, and by a combination of weak attractive orrepellent intramolecular forces involved in hydrogen bonding, ionicbonding, and hydrophobic interactions. When pairs of cysteine residuesare brought into close proximity as the peptide backbone folds, strongcovalent disulfide bonds often form between cysteine residues, servingto lock the tertiary conformation in place. Refolding protocols havebeen designed to break incorrect disulfide bonds, block random disulfidebonding, and allow refolding and correct disulfide bonding underconditions favorable to the formation of an active conformer.

One general method for recovering active protein from aggregatesinvolves solubilizing the aggregated protein in strongly denaturingsolutions and then optionally exchanging weakly denaturing solutions forthe strongly denaturing solutions (or diluting the strong denaturant),or using molecular sieve or high-speed centrifugation techniques. U.S.Pat. Nos. 4,512,922; 4,518,256; 4,511,502; and 4,511,503, incorporatedherein by reference. Such recovery methods are useful within certainmethods of the invention to prepare active peptide and proteincompositions from aggregated, or aggregation-prone, starting materials.The terms “denaturant” are broadly applied herein to include denaturantand detergent compounds that unfold proteins and/or disrupt disulfidebonds and other interactions between aggregate-prone peptides andproteins. Examples of suitable materials for use as denaturants in thiscontext include, but are not limited to, the denaturants urea andguanidine-hydrochloride, and detergents such as polyoxyethylenep-tert-octylphenol (Nonidet®P40), polyoxyethylene, p-tert-octylphenol(Triton-X-100), and sodium deoxycholate. Often, the formulations andmethods of the invention will incorporate urea as the selecteddenaturant, because it is highly soluble in aqueous solutions and it iscapable of being removed rapidly from solution by dialysis. In addition,because urea is a nonionic substance, it does not interfere with ionexchange materials that may be used in the process to removecontaminants of bacterial origin such as DNA and endotoxin. Althoughnumerous procedures are known for solubilizing aggregated inclusion bodyproteins in the presence of denaturant, clinical use of the resultantproduct requires that the denaturant contained therein be replaced withclinically acceptable materials which are nontoxic and nonirritating, sothat the resultant solution complies with medical standards forinjection into humans.

Certain aggregation inhibitory methods for use within the invention seekto eliminate random disulfide bonding prior to coaxing the recombinantprotein into its biologically active conformation. The denatured peptideor protein to be refolded is then further purified under reducingconditions that maintain the cysteine moieties of the protein as freesulfhydryl groups. The reducing agent is then diluted into an aqueoussolution to enable the refolded protein to form the appropriatedisulfide bonds in the presence of air or some other oxidizing agent.This enables refolding to be easily incorporated into the overallpurification or formulation process.

In another approach that is useful within the methods and formulationsof the invention, refolding of recombinant peptide or protein takesplace in the presence of both the reduced (R—SH) and oxidized (R—S—S—R)forms of a sulfhydryl compound. This allows free sulfhydryl groups anddisulfides to be formed and reformed constantly throughout thepurification process. The reduced and oxidized forms of the sulfhydrylcompound are provided in a buffer having sufficient denaturing powerthat all of the intermediate conformations of the protein remain solublein the course of the unfolding and refolding. Urea is a suitable buffermedium because of its apparent ability to act both as a sufficientlyweak denaturing agent to allow the protein to approximate its correctconformation, and as a sufficiently strong denaturant that the refoldingintermediates maintain their solubility.

Yet another alternative purification/preparative technique for usewithin the methods and compositions of the invention is designed tobreak any disulfide bonds that may have formed incorrectly duringisolation of peptide or protein from aggregated form, and then toderivatize the available free sulfhydryl groups of the recombinantprotein. This objective is achieved by sulfonating the protein to blockrandom disulfide pairings, allowing the protein to refold correctly inweak denaturant, and then desulfonating the protein, under conditionsthat favor correct disulfide bonding. The desulfonation takes place inthe presence of a sulfhydryl compound and a small amount of itscorresponding oxidized form to ensure that suitable disulfide bonds willremain intact. The pH is raised to a value such that the sulfhydrylcompound is at least partially in ionized form to enhance nucleophilicdisplacement of the sulfonate.

Additional recovery methods useful for providing active peptides andproteins in unaggregated form for mucosal administration according tothe invention is provided in WO 88/8003, and Halenbeck, et al.,Bio/Technology 7:710-715, 1989, each incorporated herein by reference.These procedures involve initial solubilization of monomers isolatedfrom inclusion bodies under reducing conditions in a chaotropicenvironment comprising urea or guanidine hydrochloride, followed byrefolding by stepwise dilution of the chaotropic agents, and finaloxidation of the refolded molecules in the presence of air or aredox-system.

It is also contemplated that certain aggregated peptides and proteins tobe processed and mucosally administered according to the methods of theinvention will be solubilized and sulphitolysed in denaturant, thenprecipitated by solvent exchange. U.S. Pat. No. 4,923,967; and EP361,830, each incorporated herein by reference. According to thistechnique, the precipitated protein is resolubilized in denaturant andallowed to refold in the presence of reducing agent.

Additional methods useful within the invention for refolding proteins toan active form for mucosal administration involve the use of highconcentrations of copper as an oxidant, as employed for interleukin-2(IL-2). Tsuji, et al., Biochemistry 26:3129-3134, 1987; WO 88/8849, eachincorporated herein by reference. According to another technique, adenaturing agent and reducing agent are added to solubilize the protein,followed by removal of the reducing agent, oxidation of the protein, andremoval of the denaturant, as employed for growth hormone. U.S. Pat. No.4,985,544, each incorporated herein by reference. Other methods forrefolding are disclosed in George, et al., DNA 4:273-281, 1984; Gill, etal., Bio/Technology 3:643-646, 1985; Sekine, et al., Proc. Natl. Acad.Sci. USA 82:4306-4310, 1985, each incorporated herein by reference. Yetadditional refolding methods useful within the invention are describedin Green, et al., J. Dairy Res. 52:281-286, 1985; Winkler, et al.,Bio/Technology 3:990-1000, 1985; U.S. Pat. No. 4,652,630 (urea used forsolubilization, followed by a mild oxidizing agent for refolding); EP360,937; Boss, et al., Nucl. Acids Res. 12:3791-3806, 1984; Cabilly, etal., Proc. Natl. Acad. Sci. USA 81:3273-3277, 1984; Marston et al.,Bio/Technology 2:800-804, 1984; and Marston, Biochem. J. 240:1-12, 1986,each incorporated herein by reference.

Yet additional techniques for refolding peptides and proteins to activeforms for mucosal administration involve the use of SDS forsolubilization and Cu⁺² ions as oxidation promoters of the fully reducedproteins (e.g., as exemplified for IL-2 and IFN-Beta in U.S. Pat. No.4,572,798, incorporated herein by reference). Alternative methods forpreparing active recombinant proteins from aggregates are described inU.S. Pat. No. 4,620,948, incorporated herein by reference, which involveusing strong denaturing agents to solubilize the proteins, reducingconditions to facilitate correct folding, and denaturant replacement inthe presence of air or other oxidizing agents to reform the disulfidebonds.

Alternate methods for renaturing unfolded peptides and proteins withinthe methods and compositions of the invention involve reversibly bindingthe denatured peptide or protein to a solid matrix and stepwiserenaturing it by diluting the denaturant (as exemplified for cytochromec, ovalbumin, and trypsin inhibitor in WO 86/5809, incorporated hereinby reference). Alternatively, peptides and proteins from aggregates canbe S-sulfonated during purification to protect thiol moieties and thendimerized in the presence of oxidizing agents to yield an active productas described for a modified monomeric form of human platelet-derivedgrowth factor (PDGF) expressed in E. coli by Hoppe, et al., Biochemistry28:2956-2960, 1989, incorporated herein by reference.

Additionally, EP 433,225, published Jun. 19, 1991, incorporated hereinby reference, discloses a process for producing dimeric biologicallyactive transforming growth factor-.beta. protein or a salt thereofwherein the denatured monomeric form of the protein is subjected torefolding conditions that include a solubilizing agent such as milddetergent, an organic, water-miscible solvent, and/or a phospholipid.U.S. Pat. No. 4,705,848, incorporated herein by reference, discloses theisolation of monomeric, biologically active growth hormone frominclusion bodies using one denaturing step with a guanidine salt and onerenaturing step. Bowden, et al., Bio/Technology 9:725-730, 1991;Samuelsson, et al., Bio/Technology 9:731, 1991; and Hejnaes, et al.,Protein Engineering 5:797-806, 1992, each incorporated herein byreference, describe additional procedures and reagents that are usefulto prepare and/or stabilize aggregation-prone peptides and proteinswithin the methods and compositions of the invention.

Other methods useful within the invention for resolving aggregationproblems involve disulfide exchange equilibration of refoldingintermediates. For example, the refolding of IGF-I using redox bufferswas investigated and the partially oxidized IGF-I forms produced werecharacterized by Hober, et al., Biochemistry 31:1749-1756, 1992,incorporated herein by reference. Disulfide exchange can also bemodulated using the additive agent of peptidyl disulfide isomerase (PDI)or peptidyl prolyl isomerase (PPI). See, for example, JP PatentApplication No. 63294796; EP 413,440; and EP 293,793, each incorporatedherein by reference.

Enhancement of selected disulfide pairings, e.g., by adding 50% methanolto buffer at low ionic strength, is another useful method for preparingactive peptide and protein reagents for intranasal administrationaccording to the invention. Snyder, J. Biol. Chem. 259:7468-7472, 1984,incorporated herein by reference. This method involves enhancingformation of specific disulfide bonds by adjusting electrostatic factorsin the medium to favor the juxtaposition of oppositely charged aminoacids that border the selected cysteine residues (see also, Tamura, etal., abstract and poster presented at the Eleventh American PeptideSymposium on Jul. 11, 1989, incorporated herein by reference, whichdiscloses addition of acetonitrile, DMSO, methanol, or ethanol toimprove processing of correctly folded IGF-I).

Related methods that are useful within the invention involve changingthe redox potential of a subject peptide or protein by dialysis againsta buffer containing from 20-40% v/v ethanol over a period of up to fivehours and acidifying the mixture, e.g., as disclosed for AlaGlu-IGF-I inWO 92/03477, incorporated herein by reference. Alternatively, methanolcan be used at certain concentrations in the denaturation of activepeptides and proteins. Lustig, et al., Biochim. Biophys. Acta.1119:205-210, 1992, incorporated herein by reference. Yet additionalmethods involve the use of moderate concentrations of alcohol or othermethods of modulating solution polarity to reduce association ofpeptides under conditions that promote structure destabilization.Bryant, et al., Biochemistry 31:5692-5698, 1992; Hua, et al., Biochim.Biophys. Acta. 1078:101-110, 1991; Brems, et al., Biochemistry29:9289-9293, 1990; JP 62-190199, Jackson, et al., Biochim Biophys.Acta. 1118:139-143, 1992; Shibata, et al., Biochemistry 31:5728-5733,1992; Zhong, et al., Proc. Natl. Acad. Sci. USA 89:4462-4465, 1992, eachincorporated herein by reference.

In additional methods useful within the invention, low copper ormanganese concentrations are used to facilitate disulfide oxidation ofpolypeptides. U.S. Pat. No. 5,756,672, incorporated herein by reference.The peptide or protein is first maintained in an alkaline buffercomprising a chaotropic agent and a reducing agent in amounts sufficientfor solubilization. During the refolding or processing step the subjectpeptide or protein is incubated at a concentration of about 0.1 to 15mg/mL in a buffer of pH 7-12 comprising about 5-40% (v/v) of analcoholic or polar aprotic solvent, about 0.2 to 3M of an alkalineearth, alkali metal, or ammonium salt, about 0.1 to 9M of a chaotropicagent, and about 0.01 to 15 μM of a copper or manganese salt. An oxygensource is introduced, so that refolding of the peptide or protein occursduring the incubation. The essence of this method involves the use of aspecial buffer containing a minimal concentration of copper or manganesesalt to enhance refolding of misfolded polypeptides. The use ofmanganese or copper salts as oxidation catalysts avoids the necessity ofmore expensive disulfide-exchange agents such as glutathione.Furthermore, the method avoids the possibility of producing polypeptidecontaining disulfide adducts that can result when disulfide-exchangeagents are employed.

Additional techniques useful within the methods and compositions ofinvention involve the use of a pro-sequence of a naturally occurringpolypeptide to promote folding of a biologically inactive polypeptide toits active form, (e.g., as exemplified for subtilisin in U.S. Pat. No.5,191,063, incorporated herein by reference).

The foregoing recovery, purification and preparative methods andcompositions are generally useful to prepare formulations ofaggregation-prone peptides and proteins for mucosal administration.These methods and compositions of the invention further reduceaggregation problems that occur during storage, delivery, and even afterdelivery when pharmaceutical formulations comprising aggregation-pronebiologically active agents are delivered to, or absorbed into or across,a mucosal tissue. By determining the molecular pathways that contributeto aggregation of solid peptides and proteins, rational approaches forstabilization in accordance with the foregoing teachings are readilydetermined. These approaches specifically target the particularmechanisms involved in aggregation of a selected biologically activepeptide or protein within the invention. In conjunction with thesestrategies, the methods and compositions of the invention, e.g., whichinvolve admixtures or complexes of peptides or proteins with carriers,such as polymeric matrices, maintain the level of moisture activitywithin the formulation at optimal levels to reduce peptide or proteinaggregation. This can be achieved, for example, selecting a carrier ordelivery vehicle that provides for reduced water activities. The pH ofthe microenvironment for storage and/or delivery is also controlled tominimize peptide or protein aggregation, following the application ofphysicochemical principles set forth herein.

Another approach for stabilizing solid protein formulations of theinvention is to increase the physical stability of purified, e.g.,lyophilized, protein. This will inhibit aggregation via hydrophobicinteractions as well as via covalent pathways that may increase asproteins unfold. Stabilizing formulations in this context often includepolymer-based formulations, for example a biodegradable hydrogelformulation/delivery system. As noted above, the critical role of waterin protein structure, function, and stability is well known. Typically,proteins are relatively stable in the solid state with bulk waterremoved. However, solid therapeutic protein formulations may becomehydrated upon storage at elevated humidities or during delivery from asustained release composition or device. The stability of proteinsgenerally drops with increasing hydration. Water can also play asignificant role in solid protein aggregation, for example, byincreasing protein flexibility resulting in enhanced accessibility ofreactive groups, by providing a mobile phase for reactants, and byserving as a reactant in several deleterious processes such asbeta-elimination and hydrolysis.

Protein preparations containing between about 6% to 28% water are themost unstable. Below this level, the mobility of bound water and proteininternal motions are low. Above this level, water mobility and proteinmotions approach those of full hydration. Up to a point, increasedsusceptibility toward solid-phase aggregation with increasing hydrationhas been observed in several systems. However, at higher water content,less aggregation is observed because of the dilution effect.

In accordance with these principles, an effective method for stabilizingpeptides and proteins against solid-state aggregation for mucosaldelivery is to control the water content in a solid formulation andmaintain the water activity in the formulation at optimal levels. Thislevel depends on the nature of the protein, but in general, proteinsmaintained below their “monolayer” water coverage will exhibit superiorsolid-state stability. According to current FDA requirements, anacceptable protein drug containing pharmaceutical product should exhibitless than about 10% deterioration after 2 years. Cleland, J. L. and R.Langer, in “Formulation and Delivery of Proteins and Peptides,” ACSbooks, 1994, incorporated herein by reference.

A variety of additives, diluents, bases and delivery vehicles areprovided within the invention that effectively control water content toenhance protein stability. These reagents and carrier materialseffective as anti-aggregation agents in this sense include, for example,polymers of various functionalities, such as polyethylene glycol,dextran, diethylaminoethyl dextran, and carboxymethyl cellulose, whichsignificantly increase the stability and reduce the solid-phaseaggregation of peptides and proteins admixed therewith or linkedthereto. In some instances, the activity or physical stability ofproteins can also be enhanced by various additives to aqueous solutionsof the peptide or protein drugs. For example, additives, such as polyols(including sugars), amino acids, proteins such as collagen and gelatin,and various salts may be used.

Certain additives, in particular sugars and other polyols, also impartsignificant physical stability to dry, e.g., lyophilized proteins. Theseadditives can also be used within the invention to protect the proteinsagainst aggregation not only during lyophilization but also duringstorage in the dry state. For example sucrose and Ficoll 70 (a polymerwith sucrose units) exhibit significant protection against peptide orprotein aggregation during solid-phase incubation under variousconditions. These additives may also enhance the stability of solidproteins embedded within polymer matrices.

Yet additional additives, for example sucrose, stabilize proteinsagainst solid-state aggregation in humid atmospheres at elevatedtemperatures, as may occur in certain sustained-release formulations ofthe invention. Proteins such as gelatin and collagen also serve asstabilizing or bulking agents to reduce denaturation and aggregation ofunstable proteins in this context. These additives can be incorporatedinto polymeric melt processes and compositions within the invention. Forexample, polypeptide microparticles can be prepared by simplylyophilizing or spray drying a solution containing various stabilizingadditives described above. Sustained release of unaggregated peptidesand proteins can thereby be obtained over an extended period of time.

Various additional preparative components and methods, as well asspecific formulation additives, are provided herein which yieldformulations for mucosal delivery of aggregation-prone peptides andproteins, wherein the peptide or protein is stabilized in asubstantially pure, unaggregated form. A range of components andadditives are contemplated for use within these methods andformulations. Exemplary of these anti-aggregation agents are linkeddimers of cyclodextrins (CDs), which selectively bind hydrophobic sidechains of polypeptides. Breslow, et al., J. Am. Chem. Soc.120:3536-3537; Maletic, et al., Angew. Chem. Int. Ed. Engl.35:1490-1492; each incorporated herein by reference. These CD dimershave been found to bind to hydrophobic patches of proteins in a mannerthat significantly inhibits aggregation. Leung, et al., Proc. Nat.lAcad. Sci. USA 97:5050-5053, 2000, incorporated herein by reference.This inhibition is selective with respect to both the CD dimer and theprotein involved. Such selective inhibition of protein aggregationprovides additional advantages within the intranasal delivery methodsand compositions of the invention. Additional agents for use in thiscontext include CD trimers and tetramers with varying geometriescontrolled by the linkers that specifically block aggregation ofpeptides and protein. Breslow, et al., J. Am. Chem. Soc.118:11678-11681, 1996; Breslow, et al., PNAS USA 94:11156-11158, 1997;Breslow, et al., Tetrahedron Lett., 1998, pp. 2887-2890, eachincorporated herein by reference.

Yet additional anti-aggregation agents and methods for incorporationwithin the invention involve the use of peptides and peptide mimetics toselectively block protein-protein interactions. In one aspect, thespecific binding of hydrophobic side chains reported for CD multimers isextended to proteins via the use of peptides and peptide mimetics thatsimilarly block protein aggregation. A wide range of suitable methodsand anti-aggregation agents are available for incorporation within thecompositions and procedures of the invention. Zutshi, et al., Curr.Opin. Chem. Biol. 2:62-66, 1998; Daugherty, et al., J. Am. Chem. Soc.121:4325-4333, 1999: Zutshi, et al., J. Am. Chem. Soc. 119:4841-4845,1997; Ghosh, et al., Chem. Biol. 5:439-445, 1997; Hamuro, et al., Angew.Chem. Int. Ed. Engl. 36:2680-2683, 1997; Alberg, et al., Science262:248-250, 1993; Tauton, et al., J. Am. Chem. Soc. 118:10412-10422,1996; Park, et al., J. Am. Chem. Soc. 121:8-13, 1999; Prasanna, et al.,Biochemistry 37:6883-6893, 1998; Tiley, et al., J. Am. Chem. Soc.119:7589-7590, 1997; Judice, et al., PNAS, USA 94:13426-13430, 1997;Fan, et al., J. Am. Chem. Soc. 120:8893-8894, 1998; Gamboni et al.,Biochemistry 37:12189-12194, 1998, each incorporated herein byreference. Briefly, these methods involve rational design and selectionof peptides and mimetics that effectively block interactions betweenselected biologically active peptides or proteins, whereby the selectedpeptides and mimetics significantly reduce aggregation of the activepeptides or proteins in a mucosal formulation. Anti-aggregation peptidesand mimetics thus identified are coordinately administered with, oradmixed or conjugated in a combinatorial formulation with, abiologically active peptide or protein to effectively inhibitaggregation of the active peptide or protein in a manner thatsignificantly enhances absorption and/or bioavailability of the activepeptide or protein.

Other anti-aggregation agents for use within the invention includechaperonins and analogs and mimetics of such molecules, as well asantibodies and antibody fragments that function in a similar, but oftenmore specific, manner as chaperonins to bind peptide and protein domainsand thereby block associative interactions there between. Thesemolecular chaperones were initially recognized as stress proteinsproduced in cells requiring repair. In particular, studies of heat shockon enzymes showed that molecular chaperones function not only duringcellular stress but also to chaperone the process of normal proteinfolding. Chaperonins comprise a ubiquitous family of proteins thatmediate post-translational folding and assembly of other proteins intooligomeric structures. They prevent the formation of incorrectstructures, and also act to disrupt incorrect structures that formduring these processes. The chaperones non-covalently bind to theinteractive surface of a target protein. This binding is reversed undercircumstances that favor the formation of the correct structure byfolding. Chaperones have not been shown to be specific for only oneprotein, but rather act on families of proteins that have similarstoichiometric requirements (e.g., specific structural domains that arerecognized by the chaperones). Various publications describe theselection and use of chaperoning, antibodies and antibody fragments asaggregation-blocking agents for use within the invention (see, e.g., WO93/11248; WO 93/13200; WO 94/08012; WO 94/11513; WO 94/08012; and U.S.Pat. No. 5,688,651, each incorporated herein by reference).

Additional methods for inhibiting aggregation within the inventioninclude the use of fusion proteins, as disclosed for example for IGF-IEP 130,166; U.S. Pat. No. 5,019,500; and EP 219,814, each incorporatedherein by reference. These incorporated references disclose expressionof fusion peptides of IGF-I with a protective polypeptide in bacteria.EP 264,074 discloses a two-cistronic met-IGF-I expression vector with aprotective peptide of 500-50,000 molecular weight. U.S. Pat. No.5,028,531; and Saito, et al., J. Biochem. 101:1281-1288, 1987, eachincorporated herein by reference. Other fusion techniques include fusionof IGF-I with a protective peptide from which a rop gene is cut off, EP219,814, incorporated herein by reference, in which IGF-I ismultimerized, Schulz, et al., J. Bacteriol. 169:5385-5392, 1987,incorporated herein by reference, in which IGF-I is fused withluteinizing hormone (LH) through a chemically cleavable methionyl ortryptophan residue at the linking site, Saito, et al., J. Biochem.101:123-134, 1987, incorporated herein by reference, and in which IGF-Iis fused with superoxide dismutase. EP 196,056; Niwa, et al., Ann. NYAcad. Sci. 469:31-52, 1986, incorporated herein by reference. Thesedisclosures, which teach chemical synthesis, cloning, and successfulexpression of genes for IGF-I fused to another polypeptide, aregenerally applicable to prepare a range of fusion polypeptides withother therapeutic peptides and proteins for use within the invention.

Yet additional methods for use within the methods and formulations ofthe invention involve addition of a leader sequence to the subjecttherapeutic peptide or protein to improve the fidelity of folding afterrecombinant expression. In this context, U.S. Pat. No. 5,158,875,incorporated herein by reference, describes a method for refoldingrecombinant IGF-I that involves cloning the IGF-I gene with a positivelycharged leader sequence prior to transfecting the DNA into the hostcell. The additional positive charge on the amino terminus of therecombinant IGF-I promotes correct refolding when the solubilizedprotein is stirred for 2-16 hours in denaturant solution. Followingrefolding, the leader sequence is cleaved and the active recombinantprotein is purified.

Another method for facilitating in vitro refolding of recombinantpolypeptides involves using a solubilized affinity fusion partner, forexample comprising two IgG-binding domains derived from staphylococcalprotein A. Samuelsson, et al., Bio/Technology 9:731, 1991, incorporatedherein by reference. This method uses the protein A domain as asolubilizer of misfolded and multimeric IGF-I. While this method doesnot use denaturing agents or redox chemicals, it involves the addedsteps of fusing onto the IGF-I gene a separate gene and removing thepolypeptide encoded by that gene after expression of the fusion gene.

Other techniques in peptide and protein engineering disclosed hereinwill further reduce the extent of protein aggregation and instability inmucosal delivery methods and formulations of the invention. One exampleof a useful method for peptide or protein modification in this contextis PEGylation. The stability and aggregation problems of polypeptidedrugs can be significantly improved by covalently conjugatingwater-soluble polymers such as PEG with the polypeptide. Another exampleis modification of a peptide or protein amino acid sequence in terms ofthe identity or location of one or more residues, e.g., by terminal orinternal addition, deletion or substitution (e.g., deletion of cysteineresidues or replacement by alanine or serine) to reduce aggregationpotential. The improvements in terms of stability and aggregationpotential that are achieved by these methods enables effective mucosaldelivery of a therapeutically effective polypeptide or proteincomposition within the methods of the invention.

Charge Modifying and pH Control Agents and Methods

To improve the transport characteristics of biologically active agents(including peptide YY, other active peptides and proteins, andmacromolecular and small molecule drugs) for enhanced delivery acrosshydrophobic mucosal membrane barriers, the invention also providestechniques and reagents for charge modification of selected biologicallyactive agents or delivery-enhancing agents described herein. In thisregard, the relative permeabilities of macromolecules is generally berelated to their partition coefficients. The degree of ionization ofmolecules, which is dependent on the pK_(a) of the molecule and the pHat the mucosal membrane surface, also affects permeability of themolecules. Permeation and partitioning of biologically active agents,including peptide YY and analogs of the invention, for mucosal deliverymay be facilitated by charge alteration or charge spreading of theactive agent or permeabilizing agent, which is achieved, for example, byalteration of charged functional groups, by modifying the pH of thedelivery vehicle or solution in which the active agent is delivered, orby coordinate administration of a charge- or pH-altering reagent withthe active agent.

A model compound for evaluating charge- and pH-modification methods foruse within the mucosal delivery formulations and methods of theinventions is nicotine. The charge status of this model therapeutic as afunction of pH has been investigated at various delivery sites of skinand absorptive mucosae. Nair, et al., J. Pharm. Sci. 86:257-262, 1997,incorporated herein by reference. Nicotine is a diacidic base withwell-separated pKa values (3.04 and 7.84) that allow the study ofparticular species by pH control. The dissociation of nicotine followsthe pH-partition hypothesis, so the theoretical relative proportions ofthe different charged species at any particular pH can be determined. Asan ionizable compound (pKa values of 3.04 and 7.84), nicotine insolutions of different pH values provides a model for determining theinfluence of the charge status of a molecule on permeation.

The permeation of nicotine across certain mucosal and skin surfacesfollows zero-order kinetics. The rate of permeation is dependent ondonor solution pH and increases exponentially as the pH of the deliverysolution is increased. As expected with a majority of chargedmacromolecular species for use within the invention, the permeability ofnicotine across various skin and mucosal surfaces is reportedly higherun-ionized species (NN) than for ionized species (NNH⁺, NH⁺NH⁺). It isalso reported that un-ionized nicotine molecules are more permeablethrough absorptive mucosae (nasal, buccal, sublingual, and gingival)than through skin (abdominal, dorsal, thigh, and ear pinna). Partitionstudies confirm that biomembrane permeation of nicotine follows thepH-partition theory.

Consistent with these general teachings, mucosal delivery of chargedmacromolecular species, including peptide YY and other biologicallyactive peptides and proteins, within the methods and compositions of theinvention is substantially improved when the active agent is deliveredto the mucosal surface in a substantially un-ionized, or neutral,electrical charge state.

Calculation of the isoelectric points of peptide YY and otherbiologically active peptides, proteins, and peptide analogs and mimeticsis readily undertaken to guide the selection of pH and other values formucosal formulations within the invention, which optionally delivercharged macromolecules in a substantially un-ionized state to themucosal surface or, alternatively, following mucosal delivery at atarget site of drug action. The pI of an amphoteric molecule is definedas the pH at which the net charge is zero. The variation of net chargewith pH is of importance in charge-dependent separation methods likeelectrophoresis, isoelectric focusing, chromatofocusing and ion-exchangechromatography. Thus, methods for estimating isoelectric points (pI) fornative peptides and proteins are well known and readily implementedwithin the methods and compositions of the invention. Cameselle, et al.,Biochem. Educ. 14:131-136, 1986; Skoog, et al., Trends Anal. Chem.5:82-83, 1986; Sillero, et al., Anal. Biochem. 179:319-25, 1989;Englund, et al., Biochim. Biophys. Acta. 1065:185-194, 1991; Bjellquist,et al., Electrophoresis. 14:1023-1031, 1993; Mosher, et al., J.Chromatogr. 638:155-164, 1993; Bjellqvist, et al., Electrophoresis15:529-539, 1994; Watts, et al., Electrophoresis 16:22-27, 1995, eachincorporated herein by reference.

For determining pI values of peptides and proteins for use within theinvention, net charge can be estimated, for example, by the well-knownHenderson-Hasselbalch equation. These determinations are based in parton the amino acid composition of the subject peptide or protein,yielding component pI values for specific amino acid side chains and forthe N- and C-terminal groups. The individual ionizable side chains ofeach type of amino acid are typically assumed to have pKa valuesdistributed around the projected pKa, value, simulating the situation inpolypeptides and proteins where a given type of ionizable amino acidside chain often appears in several positions in the amino acid sequenceand with various individual ionization constants, depending both on theadjacent side chains and on the three-dimensional environment in theprotein. Bjellqvist, et al., Electrophoresis 15: 529-539, 1994; Matthew,Annu. Rev. Biophys. Chem. 14:387-417, 1985, each incorporated herein byreference. By assuming a distribution of pKa values, the calculatedtitration curves will be smoothed out. The presence of other chargedgroups is also taken into account. These analyses yield a set of pKavalues, including values for amino acid residues with ionizable sidechains. Each particular type of ionizable group is assumed to have pKavalues distributed around the chosen value, thereby simulating thesituation in intact proteins and polypeptides. According to these knowncalculation methods, accurate estimates of pI values for peptides andproteins show sufficient agreement with experimental values determinedfor native proteins, over a wide pH range (3.4-11), particularly whenmore refined analyses, including such factors as charge contributions ofheme groups, sialic acid residues, etc., are taken into account.Henriksson, et al., Electrophoresis. 16:1377-1380, 1995, incorporatedherein by reference.

Thus, for polypeptides of known amino acid composition, a sufficient pIvalue estimate can be calculated by use of the ionization constant pKafor amino acid side chain groups. Where other types of ionizable groupsoccur, the charge for each such group at any given pH can also bereadily estimated. The total net charge at a selected pH is obtained bysumming up the charge for each type of ionizable group times the numberof groups. In the present study, suitable average pKa, values wereselected for the ionizable amino acid side chains, and for the terminalgroups. Additional guidance for determining pI values for polypeptidesuseful within the invention is provided, for example, by Englund, etal., Biochim. Biophys. Acta 1065:185-194, 1991; Englund, et al.,Electrophoresis 14:1307-1311, 1993; Uzcategui, et al., J. Biotechnol.19:271-286, 1991; Sims, et al., Gene 74:411-422, 1988; Cameselle, etal., Biochem. Educ. 14:131-136, 1986; Skoog, et al., Trends Anal. Chem.5:82-83, 1986; Sillero, et al., Anal. Biochem. 179:319-25, 1989;Bjellquist, et al., Electrophoresis 14:1023-1031, 1993; Mosher, et al.,J. Chromatogr. 638:155-164, 1993; Bjellqvist, et al., Electrophoresis15:529-539, 1994; Watts, et al., Electrophoresis 16: 22-27, 1995; andOda, et al., Biochemistry 33: 5275-5284, 1994, each incorporated hereinby reference. These and other teachings in the art allow forsufficiently accurate determination of charge values for peptides andproteins, and ready determination of appropriate pH values and othermodifications to mucosal formulations within the invention to facilitatedelivery of peptide and protein therapeutics in a substantiallyunionized form. Naturally, pH adjustments and other modifications toalter the charge status of a given peptide or protein therapeutic aredetermined in such a manner as to preserve substantial biologicalactivity of the subject peptide or protein within the formulation orafter delivery at a target site of action.

Certain peptide YY and other biologically active peptide and proteincomponents of mucosal formulations for use within the invention will becharge modified to yield an increase in the positive charge density ofthe peptide or protein. These modifications extend also to cationizationof peptide and protein conjugates, carriers and other delivery formsdisclosed herein. Cationization offers a convenient means of alteringthe biodistribution and transport properties of proteins andmacromolecules within the invention. Cationization is undertaken in amanner that substantially preserves the biological activity of theactive agent and limits potentially adverse side effects, includingtissue damage and toxicity. In many cases, cationized molecules havehigher organ uptake and penetration compared with non-cationized forms.Ekrami, et al., Journal of Pharmaceutical Sciences 84:456-461, 1995;Bergman, et al., Clin. Sci. 67:35-43, 1984; Triguero, et al., J. Pharm.Exp. Ther. 258:186-192, 1991. In some cases, cationized proteins canpenetrate physiological barriers considered impenetrable by the nativeproteins. For example, cationized albumin, Pardridge, et al., J. Pharm.Exp. Ther. 255:893-899, 1991, incorporated herein by reference, andcationized IgG, Triguero, et al., Proc. Nat. Acad. Sci. U.S.A.86:4761-4765, 1989, incorporated herein by reference, have beendemonstrated to bind to the brain capillary endothelium in vitro andcross the blood-brain barrier in vivo to a much greater extent thannative albumin and native IgG. Cationized proteins are also generallytaken up by the lungs to a greater extent than native proteins. Bergman,et al., Clin. Sci. 67:35-43, 1984; Triguero, et al., J. Pharm. Exp.Ther. 258:186-192, 1991; Pardridge, et al., J. Pharm. Exp. Ther.251:821-826, 1989, each incorporated herein by reference. At the tissuelevel, it has been demonstrated that cationized ferritin (CF) binds toand is transcytosed across the pulmonary endothelium, Pietra, et al.,Lab Invest. 49:54-61, 1983; Pietra, et al., Lab Invest. 59:683-691,1988, in isolated, perfused rat lungs, whereas native ferritin does notbind to the pulmonary endothelium and is only transcytosed across thisbarrier to a small degree. Bergman, et al., Clin. Sci. 67:35-43, 1984,incorporated herein by reference, demonstrated that by increasing thelevel of cationization and the charge density of human serum albumin (asmeasured by the change in the pI value of native albumin), the uptake ofcationized albumins by the lungs following iv administration in rats canbe increased. Pardridge, et al. have also demonstrated that cationizedIgG and physiologically cationic histone. Pardridge, et al., J. Pharm.Exp. Ther. 251:821-826, 1989, incorporated herein by reference, havehigher uptakes in the lungs compared with native IgG and bovine albumin,respectively. However, some studies have failed to demonstrate higherlung uptake for cationized proteins compared with native proteins. Forinstance, Pardridge, et al., J. Pharm. Exp. Ther. 255:893-899, 1991,incorporated herein by reference, and Takakura, et al., Pharm. Res.7:339-346, 1990, incorporated herein by reference, report lower lunguptake for cationized albumin compared with native albumin following ivbiodistribution studies in animals.

Degradative Enzyme Inhibitory Agents and Methods

A major drawback to effective mucosal delivery of biologically activeagents, including peptide YY, is that they may be subject to degradationby mucosal enzymes. The oral route of administration of therapeuticcompounds is particularly problematic, because in addition toproteolysis in the stomach, the high acidity of the stomach destroysmany active and inactive components of mucosal delivery formulationsbefore they reach an intended target site of drug action. Furtherimpairment of activity occurs by the action of gastric and pancreaticenzymes, and exo and endopeptidases in the intestinal brush bordermembrane, and by metabolism in the intestinal mucosa where a penetrationbarrier substantially blocks passage of the active agent across themucosa.

In addition to their susceptibility to enzymatic degradation, manytherapeutic compounds, particularly relatively low molecular weightproteins, and peptides, introduced into the circulation, are clearedquickly from mammalian subjects by the kidneys. This problem may bepartially overcome by administering large amounts of the therapeuticcompound through repeated administration. However, higher doses oftherapeutic formulations containing protein or peptide components canelicit antibodies that can bind and inactivate the protein and/orfacilitate the clearance of the protein from the subject's body.Repeated administration of the formulation containing the therapeuticprotein or peptide is essentially ineffective and can be dangerous as itcan elicit an allergic or autoimmune response.

The problem of metabolic liability of therapeutic peptides, proteins andother compounds may be addressed in part through rational drug design.However, medicinal chemists have had less success in manipulating thestructures of peptides and proteins to achieve high cell membranepermeability while still retaining pharmacological activity.Unfortunately, many of the structural features of peptides and proteins(e.g., free N-terminal amino and C-terminal carboxyl groups, and sidechain carboxyl (e.g., Asp, Glu), amino (e.g., Lys, Arg) and hydroxyl(e.g. Ser, Thr, Tyr) groups) that bestow upon the molecule affinity andspecificity for its pharmacological binding partner also bestow upon themolecule undesirable physicochemical properties (e.g., charge, hydrogenbonding potential) which limit their cell membrane permeability.Therefore, alternative strategies need to be considered for intranasalformulation and delivery of peptide and protein therapeutics.

Attempts to overcome the so-called enzymatic barrier to drug deliveryinclude the use of liposomes, Takeuchi, et al., Pharm. Res. 13:896-901,1996, incorporated herein by reference, and nanoparticles, Mathiowitz,et al., Nature 386:410-4, 1997, incorporated herein by reference, thatreportedly provide protection for incorporated insulin towards anenzymatic attack and the development of delivery systems targeting tothe colon, where the enzymatic activity is comparatively low.Rubenstein, et al., J. Control Rel. 46:59-73, 1997, incorporated hereinby reference. In addition, co-administration of protease inhibitors hasbeen reported in various studies to improve the oral bioavailability ofinsulin. Fujii, et al, J. Pharm Pharmacol. 37:545-9, 1985; Yamamoto, etal., Pharm Res. 11:1496-600, 1994; Moroshita, et al., Int. J. Pharm.78:9-16, 1992, incorporated herein by reference.

More recent research efforts in the area of protease inhibition forenhanced delivery of biotherapeutic compounds, including peptide andprotein therapeutics, has focused on covalent immobilization of enzymeinhibitors on mucoadhesive polymers used as drug carrier matrices.Bernkop-Schnurch, et al., Drug Dev. Ind. Pharm. 23:733-40, 1997;Bernkop-Schnurch, et al., J. Control. Rel. 47:113-21, 1997;Bernkop-Schnurch, et al., J. Drug Targ. 7:55-63, 1999, each incorporatedherein by reference. In conjunction with these teachings, the inventionprovides in more detailed aspects an enzyme inhibitor formulated with acommon carrier or vehicle for mucosal delivery of peptide YY and otherbiologically active peptides, analogs and mimetics, optionally to beadministered coordinately one or more additional biologically active ordelivery-enhancing agents. Optionally, the enzyme inhibitor iscovalently linked to the carrier or vehicle. In certain embodiments, thecarrier or vehicle is a biodegradable polymer, for example, abioadhesive polymer. Thus, for example, a protease inhibitor, such asBowman-Birk inhibitor (BBI), displaying an inhibitory effect towardstrypsin and {acute over (α)}-chymotrypsin, Birk Y., Int. J. Pept.Protein Res. 25:113-31, 1985, incorporated herein by reference, orelastatinal, an elastase-specific inhibitor of low molecular size, maybe covalently linked to a mucoadhesive polymer as described herein. Theresulting polymer-inhibitor conjugate exhibits substantial utility as amucosal delivery vehicle for peptides and other biologically activeagents formulated or delivered alone or in combination with otherbiologically active agents or additional delivery-enhancing agents.

Exemplary mucoadhesive polymer-enzyme inhibitor complexes that areuseful within the mucosal delivery formulations and methods of theinvention include, but are not limited to:Carboxymethylcellulose-pepstatin (with anti-pepsin activity);Poly(acrylic acid)-Bowman-Birk inhibitor (anti-chymotrypsin);Poly(acrylic acid)-chymostatin (anti-chymotrypsin); Poly(acrylicacid)-elastatinal (anti-elastase); Carboxymethylcellulose-elastatinal(anti-elastase); Polycarbophil—elastatinal (anti-elastase);Chitosan—antipain (anti-trypsin); Poly(acrylic acid)—bacitracin(anti-aminopeptidase N); Chitosan—EDTA (anti-aminopeptidase N,anti-carboxypeptidase A); Chitosan—EDTA—antipain (anti-trypsin,anti-chymotrypsin, anti-elastase). Bernkop-Schnürch, J. Control. Rel.52:1-16, 1998, incorporated herein by reference. As described in furtherdetail below, certain embodiments of the invention will optionallyincorporate a novel chitosan derivative or chemically modified form ofchitosan. One such novel derivative for use within the invention isdenoted as a β-[1→4]-2-guanidino-2-deoxy-D-glucose polymer (poly-GuD).

In recent years the use of enzyme inhibitors to overcome the enzymaticbarrier to perorally administered therapeutic peptides and proteins hasgained considerable interest. For a detailed review seeBernkop-Schnürch, A., J. Control. Rel. 52:1-16, 1998, incorporatedherein by reference. However, especially for peptide and protein drugsthat are used in long-term therapy, the co-administration of enzymeinhibitors has remained questionable because of side effects caused bythese agents. Several side effects, such as systemic intoxications, adisturbed digestion of nutritive proteins, and hypertrophy as well ashyperplasia of the pancreas based on a feedback regulation, mayaccompany enzyme inhibitor co-administration by oral delivery methods.Even if systemic toxic side effects and an intestinal mucosal damage canbe excluded, enzyme inhibitors of pancreatic proteases still have atoxic potential caused by the inhibition of these digestive enzymesthemselves. Besides a disturbed digestion of nutritive proteins, aninhibitor-induced stimulation of protease secretion caused by afeed-back regulation may be expected. Reseland, et al., Hum. Clin. Nutr.126:634-642, 1996, incorporated herein by reference. Numerous studieshave investigated this feed-back regulation with inhibitors, such asBowman-Birk inhibitor, soybean trypsin inhibitor (Kunitz trypsininhibitor) and camostat, in rats and mice. They demonstrate that thisfeed-back regulation rapidly leads to both hypertrophy and hyperplasiaof the pancreas. Moreover, a prolonged oral administration of theBowman-Birk inhibitor and soybean trypsin inhibitor leads to thedevelopment of numerous neoplastic foci, frequently progressing toinvasive carcinoma. Otsuki, et al., Pancreas 2:164-169, 1987; Melmed, etal., Biochim. Biophys. Acta 421:280-288, 1976; McGuinness, et al.,Scand. J. Gastroneterol. 17:273-277, 1982; Ge, et al., Br. J. Nutr.70:333-345, 1993, each incorporated herein by reference. A reduction oreven exclusion of this feed-back regulation might be possible by thedevelopment of drug delivery systems which keep inhibitor(s)concentrated on a restricted area of the intestine, where drugliberation and subsequent absorption takes place. For a general reviewof more recent enzyme inhibitor strategies in the context of oralpeptide drug delivery, see, e.g., Marschütz, et al., Biomaterials21:1499-1507, 2000, incorporated herein by reference.

The present invention provides coordinate administration methods and/orcombinatorial formulations directed toward coordinate administration ofa biologically active agent, including one or more peptide YY proteins,analogs and mimetics, with an enzyme inhibitor. Since a variety ofdegradative enzymes are present in the mucosal environment, theprophylactic and therapeutic compositions and methods of the inventionare readily modified to incorporate the addition or coadministration ofan enzyme inhibitor, such as a protease inhibitor, with the biologicallyactive agent (e.g., a physiologically active peptide or protein), tothereby improve bioavailability of the active agent. For example, in thecase of therapeutically active peptides and proteins, one or moreprotease inhibiting agent(s) is/are optionally combined or coordinatelyadministered in a formulation or method of the invention with one ormore inhibitors of a proteolytic enzyme. In certain embodiments, theenzyme inhibitor is admixed with or bound to a common carrier with thebiologically active agent. For example, an inhibitor of proteolyticenzymes may be incorporated in a therapeutic or prophylactic formulationof the invention to protect a biologically active protein or peptidefrom proteolysis, and thereby enhance bioavailability of the activeprotein or peptide.

Any inhibitor that inhibits the activity of an enzyme to protect thebiologically active agent(s) may be usefully employed in thecompositions and methods of the invention. Useful enzyme inhibitors forthe protection of biologically active proteins and peptides include, forexample, soybean trypsin inhibitor, pancreatic trypsin inhibitor,chymotrypsin inhibitor and trypsin and chrymotrypsin inhibitor isolatedfrom potato (solanum tuberosum L.) tubers. A combination or mixtures ofinhibitors may be employed. Additional inhibitors of proteolytic enzymesfor use within the invention include ovomucoid-enzyme, gabaxatemesylate, alpha1-antitrypsin, aprotinin, amastatin, bestatin, puromycin,bacitracin, leupepsin, alpha2-macroglobulin, pepstatin and egg white orsoybean trypsin inhibitor. These and other inhibitors can be used aloneor in combination. The inhibitor(s) may be incorporated in or bound to acarrier, e.g., a hydrophilic polymer, coated on the surface of thedosage form which is to contact the nasal mucosa, or incorporated in thesuperficial phase of the surface, in combination with the biologicallyactive agent or in a separately administered (e.g., pre-administered)formulation.

The amount of the inhibitor, e.g., of a proteolytic enzyme inhibitorthat is optionally incorporated in the compositions of the inventionwill vary depending on (a) the properties of the specific inhibitor, (b)the number of functional groups present in the molecule (which may bereacted to introduce ethylenic unsaturation necessary forcopolymerization with hydrogel forming monomers), and (c) the number oflectin groups, such as glycosides, which are present in the inhibitormolecule. It may also depend on the specific therapeutic agent that isintended to be administered. Generally speaking, a useful amount of anenzyme inhibitor is from about 0.1 mg/ml to about 50 mg/ml, often fromabout 0.2 mg/ml to about 25 mg/ml, and more commonly from about 0.5mg/ml to 5 mg/ml of the of the formulation (i.e., a separate proteaseinhibitor formulation or combined formulation with the inhibitor andbiologically active agent).

With the necessary caveat of determining and considering possible toxicand other deleterious side effects, various inhibitors of proteases maybe evaluated for use within the mucosal delivery methods andcompositions of the invention. In the case of trypsin inhibition,suitable inhibitors may be selected from, e.g., aprotinin, BBI, soybeantrypsin inhibitor, chicken ovomucoid, chicken ovoinhibitor, humanpancreatic trypsin inhibitor, camostat mesilate, flavonoid inhibitors,antipain, leupeptin, p-aminobenzamidine, AEBSF, TLCK (tosyllysinechloromethylketone), APMSF, DFP, PMSF, and poly(acrylate) derivatives.In the case of chymotrypsin inhibition, suitable inhibitors may beselected from, e.g., aprotinin, BBI, soybean trypsin inhibitor,chymostatin, benzyloxycarbonyl-Pro-Phe-CHO, FK-448, chickenovoinhibitor, sugar biphenylboronic acids complexes, DFP, PMSF,β-phenylpropionate, and poly(acrylate) derivatives. In the case ofelastase inhibition, suitable inhibitors may be selected from, e.g.,elastatinal, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK)(Ala-Ala-Pro-Val disclosed as SEQ ID NO: 800), BBI, soybean trypsininhibitor, chicken ovoinhibitor, DFP, and PMSF. Other naturallyoccurring, endogenous enzyme inhibitors for additional known degradativeenzymes present in the intranasal environment, or alternatively presentin preparative materials for production of intranasal formulations, willbe readily ascertained by those skilled in the art for incorporationwithin the methods and compositions of the invention.

Additional enzyme inhibitors for use within the invention are selectedfrom a wide range of non-protein inhibitors that vary in their degree ofpotency and toxicity Stryer, L., Biochemistry, WH Freeman and Company,NY, N.Y., 1988, incorporated herein by reference. As described infurther detail below, immobilization of these adjunct agents to matricesor other delivery vehicles, or development of chemically modifiedanalogues, may be readily implemented to reduce or even eliminate toxiceffects, when they are encountered. Among this broad group of candidateenzyme inhibitors for use within the invention are organophosphorousinhibitors, such as diisopropylfluorophosphate (DFP) andphenylmethylsulfonyl fluoride (PMSF), which are potent, irreversibleinhibitors of serine proteases (e.g., trypsin and chymotrypsin). Theadditional inhibition of acetylcholinesterase by these compounds makesthem highly toxic in uncontrolled delivery settings. Stryer, L.,Biochemistry, WH Freeman and Company, NY, N.Y., 1988, incorporatedherein by reference. Another candidate inhibitor,4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF), has an inhibitoryactivity comparable to DFP and PMSF, but it is markedly less toxic.(4-Aminophenyl)-methanesulfonyl fluoride hydrochloride (APMSF) isanother potent inhibitor of trypsin, but is toxic in uncontrolledsettings. In contrast to these inhibitors,4-(4-isopropylpiperadinocarbonyl)phenyl 1,2,3,4-tetrahydro-1-naphthoatemethanesulphonate (FK-448) is a low toxic substance, representing apotent and specific inhibitor of chymotrypsin. The co-administration ofthis compound led to an enhanced intestinal absorption of insulin inrats and dogs, resulting in a decrease in blood glucose level. Thisincreased bioavailability of insulin was found to be related to theinhibition of digestive enzymes, especially chymotrypsin. Fujii, et al.,J. Pharm. Pharmacol. 37:545-549, 1985, each incorporated herein byreference. Further representatives of this non-protein group ofinhibitor candidates, and also exhibiting low toxic risk, are camostatmesilate (N,N′-dimethylcarbamoylmethyl-p-(p′-guanidino-benzoyloxy)phenylacetatemethane-sulphonate), Yamamoto, et al., Pharm. Res. 11:1496-1500, 1994,incorporated herein by reference, and Na-glycocholate, Yamamoto, et al.,Pharm. Res. 11:1496-1500, 1994; Okagava, et al., Life Sci. 55:677-683,1994, incorporated herein by reference.

Solution or powder formulations of IFN-β administered intranasallywithout surfactants were not absorbed in rabbits. However, absorptionoccurred after the addition of surfactants (non-ionic, anionic andamphoteric). Maximum concentrations of IFN in plasma were dependent onthe surfactant used, sodium glycocholate being the most effective. Totalabsorption of IFN following nasal administration with sodiumglycocholate was 2.2% of that following intravenous administration.Maitani, et al., Drug Design and Delivery 4:109-119, 1989, incorporatedherein by reference.

Yet another type of enzyme inhibitory agent for use within the methodsand compositions of the invention are amino acids and modified aminoacids that interfere with enzymatic degradation of specific therapeuticcompounds. For use in this context, amino acids and modified amino acidsare substantially non-toxic and can be produced at a low cost. However,due to their low molecular size and good solubility, they are readilydiluted and absorbed in mucosal environments. Nevertheless, under properconditions, amino acids can act as reversible, competitive inhibitors ofprotease enzymes. McClellan, et al., Biochim. Biophys. Acta.613:160-167, 1980, incorporated herein by reference. Certain modifiedamino acids can display a much stronger inhibitory activity. A desiredmodified amino acid in this context is known as a ‘transition-state’inhibitor. The strong inhibitory activity of these compounds is based ontheir structural similarity to a substrate in its transition-stategeometry, while they are generally selected to have a much higheraffinity for the active site of an enzyme than the substrate itself.Transition-state inhibitors are reversible, competitive inhibitors.Examples of this type of inhibitor are α-aminoboronic acid derivatives,such as boro-leucine, boro-valine and boro-alanine. The boron atom inthese derivatives can form a tetrahedral boronate ion that is believedto resemble the transition state of peptides during their hydrolysis byaminopeptidases. These amino acid derivatives are potent and reversibleinhibitors of aminopeptidases and it is reported that boro-leucine ismore than 100-times more effective in enzyme inhibition than bestatinand more than 1000-times more effective than puromycin. Hussain, et al.,Pharm. Res. 6:186-189, 1989, incorporated herein by reference. Anothermodified amino acid for which a strong protease inhibitory activity hasbeen reported is N-acetylcysteine, which inhibits enzymatic activity ofaminopeptidase N. Bernkop-Schnurch, et al., Pharm. Res. 14:181-185,1997, incorporated herein by reference. This adjunct agent also displaysmucolytic properties that can be employed within the methods andcompositions of the invention to reduce the effects of the mucusdiffusion barrier. Bernkop-Schnurch, et al., Pharm. Sci. 2:361-363,1996, incorporated herein by reference.

Still other useful enzyme inhibitors for use within the coordinateadministration methods and combinatorial formulations of the inventionmay be selected from peptides and modified peptide enzyme inhibitors. Animportant representative of this class of inhibitors is the cyclicdodecapeptide, bacitracin, obtained from Bacillus licheniformis.Bacitracin A has a molecular mass of 1423 Da and shows remarkableresistance against the action of proteolytic enzymes like trypsin andpepsin. Hickey, R. J., Prog. Ind. Microbiol. 5:93-150, 1964,incorporated herein by reference. It has several biological propertiesinhibiting bacterial peptidoglycan synthesis, mammalian transglutaminaseactivity, and proteolytic enzymes such as aminopeptidase N. Because ofits protease inhibitory activity, it has been used to inhibit thedegradation of various therapeutic (poly)peptides, such as insulin,metkephamid, LH-RH, and buserelin. Yamamoto, et al., Pharm. Res.11:1496-1500, 1994; Langguth, et al., J. Pharm. Pharmacol. 46:34-40,1994; Raehs, et al., Pharm. Res. 5:689-693, 1988, each incorporatedherein by reference. Besides its inhibitory activity, bacitracin alsodisplays absorption-enhancing effects without leading to a seriousintestinal mucosal damage. Gotoh, et al., Biol. Pharm. Bull. 18:794-796,1995, incorporated herein by reference.

Nevertheless, bacitracin may not be useful in certain uncontrolleddelivery settings due to its established nephrotoxicity. To date, it hasalmost exclusively been used in veterinary medicine and as a topicalantibiotic in the treatment of infections in man. Covalent linkage ofbacitracin to a mucoadhesive polymer (carbomer) has been shown toconserve the inhibitory activity of the compound within the carriermatrix. Bernkop-Schnurch, et al., Pharm. Res. 14:181-185, 1997,incorporated herein by reference.

In addition to these types of peptides, certain dipeptides andtripeptides display weak, non-specific inhibitory activity towards someproteases. Langguth, et al., J. Pharm. Pharmacol. 46:34-40, 1994,incorporated herein by reference. By analogy with amino acids, theirinhibitory activity can be improved by chemical modifications. Forexample, phosphinic acid dipeptide analogues are also ‘transition-state’inhibitors with a strong inhibitory activity towards aminopeptidases.They have reportedly been used to stabilize nasally administered leucineenkephalin Hussain, et al., Pharm. Res. 9:626-628, 1992, eachincorporated herein by reference. Another example of a transition-stateanalogue is the modified pentapeptide pepstatin, McConnell, et al., J.Med. Chem. 34:2298-2300, 1991, incorporated herein by reference, whichis a very potent inhibitor of pepsin. Structural analysis of pepstatin,by testing the inhibitory activity of several synthetic analogues,demonstrated the major structure-function characteristics of themolecule responsible for the inhibitory activity. McConnell, et al., J.Med. Chem. 34:2298-2300, 1991, incorporated herein by reference. Similaranalytic methods can be readily applied to prepare modified amino acidand peptide analogs for blockade of selected, intranasal degradativeenzymes.

Another special type of modified peptide includes inhibitors with aterminally located aldehyde function in their structure. For example,the sequence benzyloxycarbonyl-Pro-Phe-CHO, which fulfill the knownprimary and secondary specificity requirements of chymotrypsin, has beenfound to be a potent reversible inhibitor of this target proteinase.Walker, et al., Biochem. J., 1993, pp. 321-323, incorporated herein byreference. The chemical structures of further inhibitors with aterminally located aldehyde function, e.g., antipain, leupeptin,chymostatin and elastatinal, are also known in the art, as are thestructures of other known, reversible, modified peptide inhibitors, suchas phosphoramidon, bestatin, puromycin and amastatin.

Due to their comparably high molecular mass, polypeptide proteaseinhibitors are more amenable than smaller compounds to concentrateddelivery in a drug-carrier matrix. The advantages of a slow releasecarrier system for delivery of enzyme inhibitors have been discussed byKimura, et al., Biol. Pharm. Bull. 19:897-900, 1996, incorporated hereinby reference. In this study a mucoadhesive delivery system exhibited adesired release rate of the protease inhibitor aprotinin ofapproximately 10% per hour, which was almost synchronous with therelease rate of a polypeptide drug. In vivo studies with this deliverysystem showed an improved bioavailability of the drug (id.). For thisreason, and due to their low toxicity and strong inhibitory activity,polypeptide protease inhibitors will often be selected for use withinthe methods and compositions of the invention.

Additional agents for protease inhibition within the formulations andmethods of the invention involve the use of complexing agents. Theseagents mediate enzyme inhibition by depriving the intranasal environment(or preparative or therapeutic composition) of divalent cations whichare co-factors for many proteases. For instance, the complexing agentsEDTA and DTPA as coordinately administered or combinatorially formulatedadjunct agents, in suitable concentration, will be sufficient to inhibitselected proteases to thereby enhance intranasal delivery ofbiologically active agents according to the invention. Furtherrepresentatives of this class of inhibitory agents are EGTA,1,10-phenanthroline and hydroxychinoline. Ikesue, et al., Int. J. Pharm.95:171-9, 1993; Garner, et al., Biochemistry 13:3227-3233, 1974;Sangadala, et al., J. Biol. Chem. 269:10088-10092, 1994; Mizuma, et al.,Biochim. Biophys. Acta. 1335:111-119, 1997, each incorporated herein byreference. In addition, due to their propensity to chelate divalentcations, these and other complexing agents are useful within theinvention as direct, absorption-promoting agents. Lee, V. H. L., J.Control Release 13:213-334, 1990, incorporated herein by reference.

As noted in more detail elsewhere herein, it is also contemplated to usevarious polymers, particularly mucoadhesive polymers, as enzymeinhibiting agents within the coordinate administration, multi-processingand/or combinatorial formulation methods and compositions of theinvention. For example, poly(acrylate) derivatives, such as poly(acrylicacid) and polycarbophil, can affect the activity of various proteases,including trypsin, chymotrypsin. The inhibitory effect of these polymersmay also be based on the complexation of divalent cations such as Ca²⁺and Zn²⁺. LueBen, et al., Pharm. Res. 12:1293-1298, 1995, incorporatedherein by reference. It is further contemplated that these polymers mayserve as conjugate partners or carriers for additional enzyme inhibitoryagents, as described above. For example, a chitosan-EDTA conjugate hasbeen developed and is useful within the invention that exhibits a stronginhibitory effect towards the enzymatic activity of zinc-dependentproteases. The mucoadhesive properties of polymers following covalentattachment of other enzyme inhibitors in this context are not expectedto be substantially compromised, nor is the general utility of suchpolymers as a delivery vehicle for biologically active agents within theinvention expected to be diminished. On the contrary, the reduceddistance between the delivery vehicle and mucosal surface afforded bythe mucoadhesive mechanism will minimize presystemic metabolism of theactive agent, while the covalently bound enzyme inhibitors remainconcentrated at the site of drug delivery, minimizing undesired dilutioneffects of inhibitors as well as toxic and other side effects causedthereby. In this manner, the effective amount of a coordinatelyadministered enzyme inhibitor can be reduced due to the exclusion ofdilution effects.

More recent research efforts in the area of protease inhibition forenhanced delivery of peptide and protein therapeutics has focused oncovalent immobilization of protease inhibitors on mucoadhesive polymersused as drug carrier matrices. Bernkop-Schnurch, et al., Drug Dev. Ind.Pharm. 23:733-40, 1997; Bernkop-Schnurch, et al., J. Control. Rel.47:113-21, 1997; Bernkop-Schnurch, et al., J. Drug Targ. 7:55-63, 1999,each incorporated herein by reference. In conjunction with theseteachings, the invention provides in more detailed aspects an enzymeinhibitor formulated with a common carrier or vehicle for intranasaldelivery of a biologically active agent. Optionally, the enzymeinhibitor is covalently linked to the carrier or vehicle. In certainembodiments, the carrier or vehicle is a biodegradable polymer, forexample, a bioadhesive polymer. Thus, for example, a protease inhibitor,such as Bowman-Birk inhibitor (BBI), displaying an inhibitory effecttowards trypsin and {acute over (α)}-chymotrypsin, Birk, Y., Int. J.Pept. Protein Res. 25:113-31, 1985, incorporated herein by reference, orelastatinal, an elastase-specific inhibitor of low molecular size, maybe covalently linked to a mucoadhesive polymer as described herein. Theresulting polymer-inhibitor conjugate exhibits substantial utility as anintranasal delivery vehicle for biologically active agents according tothe methods and compositions of the invention.

Exemplary mucoadhesive polymer-enzyme inhibitor complexes that areuseful within the mucosal formulations and methods of the inventioninclude, but are not limited to: Carboxymethylcellulose-pepstatin (withanti-pepsin activity); Poly(acrylic acid)-Bowman-Birk inhibitor(anti-chymotrypsin); Poly(acrylic acid)-chymostatin (anti-chymotrypsin);Poly(acrylic acid)-elastatinal (anti-elastase);Carboxymethylcellulose-elastatinal (anti-elastase);Polycarbophil—elastatinal (anti-elastase); Chitosan—antipain(anti-trypsin); Poly(acrylic acid)—bacitracin (anti-aminopeptidase N);Chitosan—EDTA (anti-aminopeptidase N, anti-carboxypeptidase A);Chitosan—EDTA—antipain (anti-trypsin, anti-chymotrypsin, anti-elastase).Bernkop-Schnürch, J. Control. Rel. 52:1-16, 1998, incorporated herein byreference.

Mucolytic and Mucus-Clearing Agents and Methods

Effective delivery of biotherapeutic agents via intranasaladministration must take into account the decreased drug transport rateacross the protective mucus lining of the nasal mucosa, in addition todrug loss due to binding to glycoproteins of the mucus layer. Normalmucus is a viscoelastic, gel-like substance consisting of water,electrolytes, mucins, macromolecules, and sloughed epithelial cells. Itserves primarily as a cytoprotective and lubricative covering for theunderlying mucosal tissues. Mucus is secreted by randomly distributedsecretory cells located in the nasal epithelium and in other mucosalepithelia. The structural unit of mucus is mucin. This glycoprotein ismainly responsible for the viscoelastic nature of mucus, although othermacromolecules may also contribute to this property. In airway mucus,such macromolecules include locally produced secretory IgA, 1 gM, IgE,lysozyme, and bronchotransferrin, which also play an important role inhost defense mechanisms.

The thickness of mucus varies from organ to organ and between species.However, mucin glycoproteins obtained from different sources havesimilar overall amino acid and protein/carbohydrate compositions,although the molecular weight may vary over a wide. Mucin consists of alarge protein core with oligosaccharide side-chains attached through theO-glycosidic linkage of galactose or N-acetyl glucosamine to hydroxylgroups of serine and threonine residues. Either sialic acid or L-fucoseforms the terminal group of the side chain oligosaccharides with sialicacid (negatively charged at pH greater than 2.8) forming 50 to 60% ofthe terminal groups. The presence of cysteine in the end regions of themucin core facilitates cross-linking of mucin molecules via disulfidebridge formation.

The presence of a mucus layer that coats all epithelial surfaces hasbeen largely overlooked in the elucidation of epithelial penetrationenhancement mechanisms to date. This is partly because the role of mucusin the absorption of peptide and protein drugs has not yet been wellestablished. However, for these and other drugs exhibiting acomparatively high molecular mass, the mucus layer covering the nasalmucosal surfaces may represent an almost insurmountable barrier.According to the conventional formula for calculation of the diffusioncoefficient, in which the radius of the molecule indirectly correlateswith the diffusion coefficient, the mucus barrier increases tremendouslyfor polypeptide drugs. Studies focusing on this so called ‘diffusionbarrier’ have demonstrated that proteins of a molecular mass greaterthan approximately 5 kDa exhibit minimal or no permeation into mucuslayers. Allen, et al., Mucus Medicine and Biology, E. N. Elder, J. B.Elstein (eds.) p. 115, Vol. 144, Plenum Press, New York, 1982;Bernkop-Schnurch., Pharm. Sci. 2:361, 1996, each incorporated herein byreference.

The coordinate administration methods of the instant inventionoptionally incorporate effective mucolytic or mucus-clearing agents,which serve to degrade, thin or clear mucus from intranasal mucosalsurfaces to facilitate absorption of intranasally administeredbiotherapeutic agents. Within these methods, a mucolytic ormucus-clearing agent is coordinately administered as an adjunct compoundto enhance intranasal delivery of the biologically active agent.Alternatively, an effective amount of a mucolytic or mucus-clearingagent is incorporated as a processing agent within a multi-processingmethod of the invention, or as an additive within a combinatorialformulation of the invention, to provide an improved formulation thatenhances intranasal delivery of biotherapeutic compounds by reducing thebarrier effects of intranasal mucus.

A variety of mucolytic or mucus-clearing agents are available forincorporation within the methods and compositions of the invention. Lee,et al., Crit. Rev. Ther. Drug Carrier Syst. 8:91-192, 1991;Bernkop-Schnurch, et al., Arzneimittelforschung 49:799-803, 1999, eachincorporated herein by reference. Based on their mechanisms of action,mucolytic and mucus clearing agents can often be classified into thefollowing groups: proteases (e.g., pronase, papain) that cleave theprotein core of mucin glycoproteins; sulfhydryl compounds that splitmucoprotein disulfide linkages; and detergents (e.g., Triton X-100,Tween 20) that break non-covalent bonds within the mucus (see, e.g.,Allen, A. in “Physiology of the Gastrointestinal Tract,” L. R. Johnson(ed.), p. 617, Raven Press, New York, 1981, incorporated herein byreference). Additional compounds in this context include, but are notlimited to, bile salts and surfactants, for example, sodiumdeoxycholate, sodium taurodeoxycholate, sodium glycocholate, andlysophosphatidylcholine.

The effectiveness of bile salts in causing structural breakdown of mucusis in the order deoxycholate>taurocholate>glycocholate. Other effectiveagents that reduce mucus viscosity or adhesion to enhance intranasaldelivery according to the methods of the invention include, e.g.,short-chain fatty acids, and mucolytic agents that work by chelation,such as N-acylcollagen peptides, bile acids, and saponins (the latterfunction in part by chelating Ca²⁺ and/or Mg²⁺ which play an importantrole in maintaining mucus layer structure).

Additional mucolytic agents for use within the methods and compositionsof the invention include N-acetyl-L-cysteine (ACS), a potent mucolyticagent that reduces both the viscosity and adherence of bronchopulmonarymucus and is reported to modestly increase nasal bioavailability ofhuman growth hormone in anesthetized rats (from 7.5 to 12.2%). O'Hagen,et al., Pharm. Res. 7:772, 1990, incorporated herein by reference. Theseand other mucolytic or mucus-clearing agents are contacted with thenasal mucosa, typically in a concentration range of about 0.2 to 20 mM,coordinately with administration of the biologically active agent, toreduce the polar viscosity and/or elasticity of intranasal mucus.

Still other mucolytic or mucus-clearing agents may be selected from arange of glycosidase enzymes, which are able to cleave glycosidic bondswithin the mucus glycoprotein. α-amylase and β-amylase arerepresentative of this class of enzymes, although their mucolytic effectmay be limited. Leiberman, J., Am. Rev. Respir. Dis., 97:662, 1967,incorporated herein by reference. In contrast, bacterial glycosidaseswhich allow these microorganisms to permeate mucus layers of theirhosts, Corfield, et al, Glycoconjugate J. 10:72, 1993, incorporatedherein by reference, are highly mucolytic active.

For selecting mucolytic agents for use within the methods andcompositions of the invention, it is important to consider the chemicalnature of both the mucolytic (or mucus-clearing) and biologically activeagents. For example, the proteolytic enzyme pronase exhibits a verystrong mucolytic activity at pH 5.0, as well as at pH 7.2. In contrast,the protease papain exhibited substantial mucolytic activity at pH 5.0,but no detectable mucolytic activity at pH 7.2. The reason for thesedifferences in activity are explained in part by the distinct pH-optimumfor papain, reported to be pH 5. Karlson, P., Biochemie, Thieme, Verlag,Stuttgart, New York, 1984, incorporated herein by reference. Thus,mucolytic and other enzymes for use within the invention are typicallydelivered in formulations having a pH at or near the pH optimum of thesubject enzyme.

With respect to chemical characterization of the biologically activeagent, one notable concern is the vulnerability of peptide and proteinmolecules to the degradative activities of proteases and sulfhydryl. Inparticular, peptide and protein drugs can be attacked by different typesof mucolytic agents. In one study, the mucolytic proteases pronase andpapain (which each are endopeptidases that cleave at a high number ofbonds) were shown to completely degrade insulin within 2-3 h at pH 7.2.Bernkop-Schnurch, et al., Arzneimittelforschung 49:799-803, 1999,incorporated herein by reference. In contrast, at pH 2.5 insulin was notat all, or only slightly, degraded by pronase and papain, which can beexplained by the pH optimum of both enzymes being far away from pH 2.5.Whereas pronase represents an unusually non-specific protease, papaincleaves after Arg, Lys, Leu, and Gly, Karlson, P., Biochemie, Thieme,Verlag, Stuttgart, New York, 1984, incorporated herein by reference,which are all included in the primary structure of insulin and serve asan additional guide to selection of mucolytic and mucus-clearing agentswithin the invention.

The presence and number of cysteine residues and disulfide bonds inpeptide and protein therapeutics are also important factors to considerin selecting mucolytic or mucus-clearing agents within the invention.When insulin, which displays three disulfide bonds within its molecularstructure, is incubated with di-thiothreitol or N-acetylcysteine, thereis a rapid degradation of the insulin polypeptide at pH 7.2. Asubstantially lower degree of degradation at pH 2.5 is attributed to therelatively low amount of reactive thiolate anions (responsible fornucleophilic attack on disulfide bonds) at this pH value.Bernkop-Schnurch, et al., Arzneimittelforschung 49:799-803, 1999,incorporated herein by reference.

Whereas, it is generally contraindicated to use general proteases suchas pronase or papain in combination with peptide or protein drugs, thepractical use of more specific proteases can be undertaken according tothe above principals, as can the use of sulfhydryl compounds. Fortherapeutic polypeptides that exhibit no cysteine moieties within theirprimary structure (e.g. cyclosporin), the use of sulfhydryl compounds isnot problematic. Moreover, even for protein drugs bearing disulfidebonds the use of sulfhydryl compounds can be achieved, particularlywhere the disulfide bonds are not accessible for thiol attack due to theconformation of the protein, they should remain stable in the presenceof this type of mucolytic agents.

For combinatorial use with most biologically active agents within theinvention, including peptide and protein therapeutics, non-ionogenicdetergents are generally also useful as mucolytic or mucus-clearingagents. These agents typically will not modify or substantially impairthe activity of therapeutic polypeptides.

Ciliostatic Agents and Methods

Because the self-cleaning capacity of certain mucosal tissues (e.g.,nasal mucosal tissues) by mucociliary clearance is necessary as aprotective function (e.g., to remove dust, allergens, and bacteria), ithas been generally considered that this function should not besubstantially impaired by mucosal medications. Mucociliary transport inthe respiratory tract is a particularly important defense mechanismagainst infections. Wasserman., J. Allergy Clin. Immunol. 73:17-19,1984, incorporated herein by reference. To achieve this function,ciliary beating in the nasal and airway passages moves a layer of mucusalong the mucosa to removing inhaled particles and microorganisms.During chronic bronchitis and chronic sinusitis, tracheal and nasalmucociliary clearance are often impaired. Wanner, Am. Rev. Respir. Dis.116:73-125, 1977, incorporated herein by reference. This is presumablydue to either excess secretion, Dulfano, et al., Am. Rev. Respir. Dis.104:88-98, 1971, increased viscosity of mucus, Chen, et al., J. Lab.Clin. Med. 91:423-431, 1978, incorporated herein by reference,alterations in ciliary activity caused by decreased beat frequency,Puchelle, et al., Biorheology 21:265-272, 1984, incorporated herein byreference), loss of portions of the ciliated epithelium, Chodosh, etal., Am. Rev. Respir. Dis. 104:888-898, 1971, incorporated herein byreference, or to a combination of these factors. Decreased clearancepresumably favors bacterial colonization of respiratory mucosalsurfaces, predisposing the subject to infection. The ability tointerfere with this host defense system may contribute significantly toa pathological organism's virulence.

Various reports show that mucociliary clearance can be impaired bymucosally administered drugs, as well as by a wide range of formulationadditives including penetration enhancers and preservatives. Forexample, ethanol at concentrations greater than 2% has been shown toreduce the in vitro ciliary beating frequency. This may be mediated inpart by an increase in membrane permeability that indirectly enhancesflux of calcium ion which, at high concentration, is ciliostatic, or bya direct effect on the ciliary axoneme or actuation of regulatoryproteins involved in a ciliary arrest response. Exemplary preservatives(methyl-p-hydroxybenzoate (0.02% and 0.15%), propyl-p-hydroxybenzoate(0.02%), and chlorobutanol (0.5%)) reversibly inhibit ciliary activityin a frog palate model. Other common additives (EDTA (0.1%),benzalkoniuin chloride (0.01%), chlorhexidine (0.01%), phenylinercuricnitrate (0.002%), and phenylmercuric borate (0.002%), have been reportedto inhibit mucociliary transport irreversibly. In addition, severalpenetration enhancers including STDHF, laureth-9, deoxycholate,deoxycholic acid, taurocholic acid, and glycocholic acid have beenreported to inhibit ciliary activity in model systems.

Despite the potential for adverse effects on mucociliary clearanceattributed to ciliostatic factors, ciliostatic agents nonetheless finduse within the methods and compositions of the invention to increase theresidence time of mucosally (e.g., intranasally) administered peptide YYproteins, analogs and mimetics, and other biologically active agentsdisclosed herein. In particular, the delivery these agents within themethods and compositions of the invention is significantly enhanced incertain aspects by the coordinate administration or combinatorialformulation of one or more ciliostatic agents that function toreversibly inhibit ciliary activity of mucosal cells, to provide for atemporary, reversible increase in the residence time of the mucosallyadministered active agent(s). For use within these aspects of theinvention, the foregoing ciliostatic factors, either specific orindirect in their activity, are all candidates for successful employmentas ciliostatic agents in appropriate amounts (depending onconcentration, duration and mode of delivery) such that they yield atransient (i.e., reversible) reduction or cessation of mucociliaryclearance at a mucosal site of administration to enhance delivery ofpeptide YY proteins, analogs and mimetics, and other biologically activeagents disclosed herein, without unacceptable adverse side effects.

Within more detailed aspects, a specific ciliostatic factor is employedin a combined formulation or coordinate administration protocol with oneor more peptide YY proteins, analogs and mimetics, and/or otherbiologically active agents disclosed herein. Various bacterialciliostatic factors isolated and characterized in the literature may beemployed within these embodiments of the invention. For example,Hingley, et al., Infection and Immunity. 51:254-262, 1986, incorporatedherein by reference, have recently identified ciliostatic factors fromthe bacterium Pseudomonas aeruginosa. These are heat-stable factorsreleased by Pseudomonas aeruginosa in culture supernatants that havebeen shown to inhibit ciliary function in epithelial cell cultures.Exemplary among these cilioinhibitory components are a phenazinederivative, a pyo compound (2-alkyl-4-hydroxyquinolines), and arhamnolipid (also known as a hemolysin). Inhibitory concentrations ofthese and other active components were established by quantitativemeasures of ciliary motility and beat frequency. The pyo compoundproduced ciliostasis at concentrations of 50 μg/ml and without obviousultrastructural lesions. The phenazine derivative also inhibited ciliarymotility but caused some membrane disruption, although at substantiallygreater concentrations of 400 μg/ml. Limited exposure of trachealexplants to the rhamnolipid resulted in ciliostasis which was associatedwith altered ciliary membranes. More extensive exposure to rhamnolipidwas associated with removal of dynein arms from axonemes. It is proposedthat these and other bacterial ciliostatic factors have evolved toenable P. aeruginosa to more easily and successfully colonize therespiratory tract of mammalian hosts. On this basis, respiratorybacteria are useful pathogens for identification of suitable, specificciliostatic factors for use within the methods and compositions of theinvention.

Several methods are available to measure mucociliary clearance forevaluating the effects and uses of ciliostatic agents within the methodsand compositions of the invention. Nasal mucociliary clearance can bemeasured by monitoring the disappearance of visible tracers such asIndia ink, edicol orange powder, and edicol supra orange. These tracersare followed either by direct observation or with the aid of posteriorrhinoscopy or a binocular operating microscope. This method simplymeasures the time taken by a tracer to travel a definite distance. Inmore modern techniques, radiolabeled tracers are administered as anaerosol and traced by suitably collimated detectors. Alternatively,particles with a strong taste like saccharin can be placed in the nasalpassage and assayed to determine the time before the subject firstperceives the taste is used as an indicator of mucociliary clearance.

Additional assays are known in the art for measuring ciliary beatactivity. For example, a laser light scattering technique to measuretracheobronchial mucociliary activity is based on mono-chromaticity,coherence, and directionality of laser light. Ciliary motion is measuredas intensity fluctuations due to the interference of Doppler-shiftedscattered light. The scattered light from moving cilia is detected by aphotomultiplier tube and its frequency content analyzed by a signalcorrelator yielding an autocorrelation function of the detectedphotocurrents. In this way, both the frequency and synchrony of beatingcilia can be measured continuously. Through fiberoptic rhinoscopy, thismethod also allows the measurement of ciliary activity in the peripheralparts of the nasal passages.

In vitro assays for evaluating ciliostatic activity of formulationswithin the invention are also available. For example, a commonly usedand accepted assay in this context is a rabbit tracheal explant system.Gabridge, et al., Pediatr. Res. 1:31-35, 1979; Chandler, et al., Infect.Immun. 29:1111-1116, 1980, each incorporated herein by reference. Otherassay systems measure the ciliary beat frequency of a single cell or asmall number of cells. Kennedy, et al., Exp. Cell Res. 135:147-156,1981; Rutland, et al., Lancet ii: 564-565, 1980; Verdugo, et al.,Pediatr. Res. 13:131-135, 1979, each incorporated herein by reference.

Surface Active Agents and Methods

Within more detailed aspects of the invention, one or more membranepenetration-enhancing agents may be employed within a mucosal deliverymethod or formulation of the invention to enhance mucosal delivery ofpeptide YY proteins, analogs and mimetics, and other biologically activeagents disclosed herein. Membrane penetration enhancing agents in thiscontext can be selected from: (i) a surfactant, (ii) a bile salt, (iii)a phospholipid additive, mixed micelle, liposome, or carrier, (iv) analcohol, (v) an enamine, (vi) an NO donor compound, (vii) a long-chainamphipathic molecule (viii) a small hydrophobic penetration enhancer;(ix) sodium or a salicylic acid derivative; (x) a glycerol ester ofacetoacetic acid, (xi) a cyclodextrin or beta-cyclodextrin derivative,(xii) a medium-chain fatty acid, (xiii) a chelating agent, (xiv) anamino acid or salt thereof, (xv) an N-acetylamino acid or salt thereof,(xvi) an enzyme degradative to a selected membrane component, (xvii) aninhibitor of fatty acid synthesis, or (xviii) an inhibitor ofcholesterol synthesis; or (xix) any combination of the membranepenetration enhancing agents recited in (i)-(xviii).

Certain surface-active agents are readily incorporated within themucosal delivery formulations and methods of the invention as mucosalabsorption enhancing agents. These agents, which may be coordinatelyadministered or combinatorially formulated with peptide YY proteins,analogs and mimetics, and other biologically active agents disclosedherein, may be selected from a broad assemblage of known surfactants.Surfactants, which generally fall into three classes: (1) nonionicpolyoxyethylene ethers; (2) bile salts such as sodium glycocholate (SGC)and deoxycholate (DOC); and (3) derivatives of fusidic acid such assodium taurodihydrofusidate (STDHF). The mechanisms of action of thesevarious classes of surface active agents typically includesolubilization of the biologically active agent. For proteins andpeptides which often form aggregates, the surface active properties ofthese absorption promoters can allow interactions with proteins suchthat smaller units such as surfactant coated monomers may be morereadily maintained in solution. These monomers are presumably moretransportable units than aggregates. A second potential mechanism is theprotection of the peptide or protein from proteolytic degradation byproteases in the mucosal environment. Both bile salts and some fusidicacid derivatives reportedly inhibit proteolytic degradation of proteinsby nasal homogenates at concentrations less than or equivalent to thoserequired to enhance protein absorption. This protease inhibition may beespecially important for peptides with short biological half-lives.

Degradation Enzymes and Inhibitors of Fatty Acid and CholesterolSynthesis

In related aspects of the invention, peptide YY proteins, analogs andmimetics, and other biologically active agents for mucosaladministration are formulated or coordinately administered with apenetration enhancing agent selected from a degradation enzyme, or ametabolic stimulatory agent or inhibitor of synthesis of fatty acids,sterols or other selected epithelial barrier components. U.S. Pat. No.6,190,894, incorporated herein by reference. In one embodiment, knownenzymes that act on mucosal tissue components to enhance permeabilityare incorporated in a combinatorial formulation or coordinateadministration method of instant invention, as processing agents withinthe multi-processing methods of the invention. For example, degradativeenzymes such as phospholipase, hyaluronidase, neuraminidase, andchondroitinase may be employed to enhance mucosal penetration of peptideYY proteins, analogs and mimetics, and other biologically active agents,Squier, Brit. J. Dermatol. 111:253-264, 1984; Aungst and Rogers, Int. J.Pharm. 53:227-235, 1989, incorporated herein by reference, withoutcausing irreversible damage to the mucosal barrier. In one embodiment,chondroitinase is employed within a method or composition as providedherein to alter glycoprotein or glycolipid constituents of thepermeability barrier of the mucosa, thereby enhancing mucosal absorptionof peptide YY proteins, analogs and mimetics, and other biologicallyactive agents disclosed herein.

With regard to inhibitors of synthesis of mucosal barrier constituents,it is noted that free fatty acids account for 20-25% of epitheliallipids by weight. Two rate limiting enzymes in the biosynthesis of freefatty acids are acetyl CoA carboxylase and fatty acid synthetase.Through a series of steps, free fatty acids are metabolized intophospholipids. Thus, inhibitors of free fatty acid synthesis andmetabolism for use within the methods and compositions of the inventioninclude, but are not limited to, inhibitors of acetyl CoA carboxylasesuch as 5-tetradecyloxy-2-furancarboxylic acid (TOFA); inhibitors offatty acid synthetase; inhibitors of phospholipase A such as gomisin A,2-(p-amylcinnamyl)amino-4-chlorobenzoic acid, bromophenacyl bromide,monoalide, 7,7-dimethyl-5,8-eicosadienoic acid, nicergoline,cepharanthine, nicardipine, quercetin, dibutyryl-cyclic AMP, R-24571,N-oleoylethanolamine, N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphostidylserine, cyclosporine A, topical anesthetics, including dibucaine,prenylamine, retinoids, such as all-trans and 13-cis-retinoic acid, W-7,trifluoperazine, R-24571 (calmidazolium), 1-hexadocyl-3-trifluoroethylglycero-sn-2-phosphomenthol (MJ33); calcium channel blockers includingnicardipine, verapamil, diltiazem, nifedipine, and nimodipine;antimalarials including quinacrine, mepacrine, chloroquine andhydroxychloroquine; beta blockers including propanalol and labetalol;calmodulin antagonists; EGTA; thimersol; glucocorticosteroids includingdexamethasone and prednisolone; and nonsteroidal antiinflammatory agentsincluding indomethacin and naproxen.

Free sterols, primarily cholesterol, account for 20-25% of theepithelial lipids by weight. The rate limiting enzyme in thebiosynthesis of cholesterol is 3-hydroxy-3-methylglutaryl (HMG) CoAreductase. Inhibitors of cholesterol synthesis for use within themethods and compositions of the invention include, but are not limitedto, competitive inhibitors of (HMG) CoA reductase, such as simvastatin,lovastatin, fluindostatin (fluvastatin), pravastatin, mevastatin, aswell as other HMG CoA reductase inhibitors, such as cholesterol oleate,cholesterol sulfate and phosphate, and oxygenated sterols, such as25-OH— and 26-OH— cholesterol; inhibitors of squalene synthetase;inhibitors of squalene epoxidase; inhibitors of DELTA7 or DELTA24reductases such as 22,25-diazacholesterol, 20,25-diazacholestenol,AY9944, and triparanol.

Each of the inhibitors of fatty acid synthesis or the sterol synthesisinhibitors may be coordinately administered or combinatoriallyformulated with one or more peptide YY proteins, analogs and mimetics,and other biologically active agents disclosed herein to achieveenhanced epithelial penetration of the active agent(s). An effectiveconcentration range for the sterol inhibitor in a therapeutic or adjunctformulation for mucosal delivery is generally from about 0.0001% toabout 20% by weight of the total, more typically from about 0.01% toabout 5%.

Nitric Oxide Donor Agents and Methods

Within other related aspects of the invention, a nitric oxide (NO) donoris selected as a membrane penetration-enhancing agent to enhance mucosaldelivery of one or more peptide YY proteins, analogs and mimetics, andother biologically active agents disclosed herein. Recently, Salzman, etal., Am. J. Physiol. 268:G361-G373, 1995, incorporated herein byreference, reported that NO donors increase the permeability ofwater-soluble compounds across Caco-2 cell monolayers with neither lossof cell viability nor lactate dehydrogenase (LDH) release. In addition,Utoguchi, et al., Pharm. Res. 15:870-876, 1998, incorporated herein byreference, demonstrated that the rectal absorption of insulin wasremarkably enhanced in the presence of NO donors, with attendant lowcytotoxicity as evaluated by the cell detachment and LDH release studiesin Caco-2 cells.

Various NO donors are known in the art and are useful in effectiveconcentrations within the methods and formulations of the invention.Exemplary NO donors include, but are not limited to, nitroglycerine,nitropruside, NOC5[3-(2-hydroxy-1-(methyl-ethyl)-2-nitrosohydrazino)-1-propanamine], NOC12[N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine], SNAP[S-nitroso-N-acetyl-DL-penicillamine], NORI and NOR4. Efficacy of theseand other NO donors, as well as other mucosal delivery-enhancing agentsdisclosed herein, for enhancing mucosal delivery of peptide YY proteins,analogs and mimetics, and other biologically active agents can beevaluated routinely according to known efficacy and cytotoxicity assaymethods (e.g., involving control coadministration of an NO scavenger,such as carboxy-PIIO) as described by Utoguchi, et al., Pharm. Res.15:870-876, 1998, incorporated herein by reference.

Within the methods and compositions of the invention, an effectiveamount of a selected NO donor is coordinately administered orcombinatorially formulated with one or more peptide YY proteins, analogsand mimetics, and/or other biologically active agents disclosed herein,into or through the mucosal epithelium.

Agents for Modulating Epithelial Junction Structure and/or Physiology

The present invention provides novel pharmaceutical compositions thatinclude a biologically active agent and a permeabilizing agent effectiveto enhance mucosal delivery of the biologically active agent in amammalian subject. The permeabilizing agent reversibly enhances mucosalepithelial paracellular transport, typically by modulating epithelialjunctional structure and/or physiology at a mucosal epithelial surfacein the subject. This effect typically involves inhibition by thepermeabilizing agent of homotypic or heterotypic binding betweenepithelial membrane adhesive proteins of neighboring epithelial cells.Target proteins for this blockade of homotypic or heterotypic bindingcan be selected from various related junctional adhesion molecules(JAMs), occludins, or claudins.

In more detailed embodiments of the invention, the permeabilizing agentis a peptide or peptide analog or mimetic. Exemplary permeabilizingpeptides comprise from about 4-25 contiguous amino acids of anextracellular domain of a mammalian JAM-1, JAM-2, or JAM-3 protein.Alternatively, the permeabilizing peptide may comprise from about 6-15contiguous amino acids of an extracellular domain of a mammalian JAM-1,JAM-2, or JAM-3 protein. In additional embodiments, the permeabilizingpeptide comprises from about 4-25 contiguous amino acids of anextracellular domain of a mammalian JAM-1, JAM-2, or JAM-3 protein, or asequence of amino acids that exhibits at least 85% amino acid identitywith a corresponding reference sequence of 4-25 contiguous amino acidsof an extracellular domain of a mammalian JAM-1, JAM-2, or JAM-3protein. In certain embodiments, the amino acid sequence of thepermeabilizing peptide exhibits one or more amino acid substitutions,insertions, or deletions compared to the corresponding referencesequence of the mammalian JAM-1, JAM-2, or JAM-3 protein. For example,the permeabilizing peptide may exhibit one or more conservative aminoacid substitutions compared to a corresponding reference sequence of amammalian JAM-1, JAM-2, or JAM-3 protein. Such functional peptideanalogs or variants may, for instance, have one or more amino acidmutations in comparison to a corresponding wild-type sequence of thesame human JAM protein (e.g., human JAM-1), wherein the mutation(s)correspond to a divergent amino acid residue or sequence identified in adifferent human JAM protein (e.g., human JAM-2 or JAM-3) or in ahomologous JAM protein found in a different species (e.g. murine, rat,or bovine JAM-1, JAM-2 or JAM-3 protein).

In more detailed embodiments, the methods and compositions of theinvention incorporate a permeabilizing peptide that is between about4-25 amino acids in length, and includes one or more contiguous sequenceelements selected from: V R (I, V, A) P, (SEQ ID NO: 1); (V, A, I) K L(S, T) C A Y, (SEQ ID NO: 2); or E D (T, S) G T Y (T, R) C (M, E), (SEQID NO: 3). In one such embodiment, the peptide will include aconservative sequence motif V R (I, V, A) P, (SEQ ID NO: 1), wherein thethird position of the motif may be represented by one of the alternativeamino acid residues I, V, or A. In another such embodiment, the peptidewill include a conservative sequence motif (V, A, I) K L (S, T) C A Y,(SEQ ID NO: 2), wherein the first position of the motif may berepresented by one of the alternative amino acid residues V, A, or I,and the fourth position of the motif may be represented by one of thealternative amino acid residues S or T. In yet another such embodiment,the peptide will include a conservative sequence motif E D (T, S) G T Y(T, R) C (M, E), (SEQ ID NO: 3), wherein the third position of the motifmay be represented by one of the alternative amino acid residues T or S,the seventh position of the motif may be represented by one of thealternative amino acid residues T or R, and the ninth position of themotif may be represented by one of the alternative residues M or E. Inexemplary embodiments, the permeabilizing peptide is between about 4-25amino acids in length and includes one or more contiguous sequenceelements selected from wild-type human JAM-1 peptide sequences VRIP,(SEQ ID NO: 4), VKLSCAY, (SEQ ID NO: 5), TGITFKSVT, (SEQ ID NO: 6),ITAS, (SEQ ID NO: 7), SVTR, (SEQ ID NO: 8), EDTGTYTCM, (SEQ ID NO: 9),and/or GFSSPRVEW, (SEQ ID NO: 10).

Within additional aspects of the invention, pharmaceutical compositionsand methods are provided which employ a permeabilizing peptidecomprising from about 4-25 contiguous amino acids of an extracellulardomain of a mammalian occludin protein. In alternate embodiments, thepermeabilizing peptide comprises from about 6-15 contiguous amino acidsof an extracellular domain of a mammalian occludin protein. In certainaspects, the permeabilizing peptide comprises from about 4-25 contiguousamino acids of an extracellular domain of a mammalian occludin proteinor comprises an amino acid sequence that exhibits at least 85% aminoacid identity with a corresponding reference sequence of 4-25 contiguousamino acids of an extracellular domain of a mammalian occludin protein.In exemplary embodiments, the permeabilizing peptide exhibits one ormore amino acid substitutions, insertions, or deletions compared to acorresponding reference sequence of the mammalian occludin protein.Often, such peptide “analogs” will exhibit one or more conservativeamino acid substitutions compared to the corresponding referencesequence of the mammalian occludin protein. In related embodiments, thepermeabilizing peptide is a human occludin peptide and the amino acidsequence of the permeabilizing peptide exhibits one or more amino acidmutations in comparison to a corresponding wild-type sequence of thesame human occludin protein, wherein the mutation(s) correspond to astructural feature (e.g., a divergent, aligned residue or sequence ofresidues) identified in a different human occludin protein or ahomologous occludin protein found in a different species.

Within other aspects of the invention, pharmaceutical compositions andmethods are provided which employ a permeabilizing peptide comprisingfrom about 4-25 contiguous amino acids of an extracellular domain of amammalian claudin protein. In alternate embodiments, the permeabilizingpeptide comprises from about 6-15 contiguous amino acids of anextracellular domain of a mammalian claudin protein. In certain aspects,the permeabilizing peptide comprises from about 4-25 contiguous aminoacids of an extracellular domain of a mammalian claudin protein orcomprises an amino acid sequence that exhibits at least 85% amino acididentity with a corresponding reference sequence of 4-25 contiguousamino acids of an extracellular domain of a mammalian claudin protein.In exemplary embodiments, the permeabilizing peptide exhibits one ormore amino acid substitutions, insertions, or deletions compared to acorresponding reference sequence of the mammalian claudin protein.Often, such peptide “analogs” will exhibit one or more conservativeamino acid substitutions compared to the corresponding referencesequence of the mammalian claudin protein. In related embodiments, thepermeabilizing peptide is a human claudin peptide and the amino acidsequence of the permeabilizing peptide exhibits one or more amino acidmutations in comparison to a corresponding wild-type sequence of thesame human claudin protein, wherein the mutation(s) correspond to astructural feature (e.g., a divergent, aligned residue or sequence ofresidues) identified in a different human claudin protein or ahomologous claudin protein found in a different species.

In yet additional embodiments, the invention provides methods andpharmaceutical compositions which employ a permeabilizing agent asdescribed above, such as a permeabilizing peptide, and one or moretherapeutic protein(s) or peptide(s) that is/are effective as ahematopoietic agent, cytokine agent, antiinfective agent, antidementiaagent, antiviral agent, antitumoral agent, antipyretic agent, analgesicagent, antiinflammatory agent, antiulcer agent, antiallergic agent,antidepressant agent, psychotropic agent, cardiotonic agent,antiarrythmic agent, vasodilator agent, antihypertensive agent,antidiabetic agent, anticoagulant agent, cholesterol-lowering agent,hormone agent, anti-osteoporosis agent, antibiotic agent, vaccine agent,and/or bacterial toxoid.

In certain embodiments of the invention, a biologically active agent anda permeabilizing agent as described above are administered incombination with one or more mucosal delivery-enhancing agent(s). Inmore detailed embodiments of the inventions, the pharmaceuticalcompositions noted above are formulated for intranasal administration.In exemplary embodiments, the formulations are provided as an intranasalspray or powder. To enhance intranasal administration, theseformulations may combine the biologically active agent andpermeabilizing agent with one or more intranasal delivery-enhancingagents selected from:

-   -   (a) an aggregation inhibitory agent;    -   (b) a charge modifying agent;    -   (c) a pH control agent;    -   (d) a degradative enzyme inhibitory agent;    -   (e) a mucolytic or mucus clearing agent;    -   (f) a ciliostatic agent;    -   (g) a membrane penetration-enhancing agent selected from (i) a        surfactant, (ii) a bile salt, (iii) a phospholipid additive,        mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an        enamine, (vi) an NO donor compound, (vii) a long-chain        amphipathic molecule (viii) a small hydrophobic penetration        enhancer; (ix) sodium or a salicylic acid derivative; (x) a        glycerol ester of acetoacetic acid, (xi) a cyclodextrin or        beta-cyclodextrin derivative, (xii) a medium-chain fatty        acid, (xiii) a chelating agent, (xiv) an amino acid or salt        thereof, (xv) an N-acetylamino acid or salt thereof, (xvi) an        enzyme degradative to a selected membrane component, (xvii) an        inhibitor of fatty acid synthesis, or (xviii) an inhibitor of        cholesterol synthesis; or (xix) any combination of the membrane        penetration enhancing agents recited in (i)-(xviii);    -   (h) a second modulatory agent of epithelial junction physiology;    -   (i) a vasodilator agent;    -   (j) a selective transport-enhancing agent; and    -   (k) a stabilizing delivery vehicle, carrier, support or        complex-forming species with which the biologically active agent        is effectively combined, associated, contained, encapsulated or        bound resulting in stabilization of the active agent for        enhanced intranasal delivery, wherein the one or more intranasal        delivery-enhancing agents comprises any one or combination of        two or more of the intranasal delivery-enhancing agents recited        in (a)-(k), and wherein the formulation of the biologically        active agent with the one or more intranasal delivery-enhancing        agents provides for increased bioavailability of the        biologically active agent delivered to a nasal mucosal surface        of a mammalian subject.

In other related aspects of the invention, the pharmaceuticalcompositions comprising a permeabilizing agent, e.g., a permeabilizingpeptide, and a biologically active agent are effective following mucosaladministration to a mammalian subject to yield enhanced bioavailabilityof the therapeutic compound, for example by yielding a peakconcentration (C_(max)) of the biologically active agent in a bloodplasma or cerebral spinal fluid (CNS) of the subject that is about 25%or greater as compared to a peak concentration of the biologicallyactive agent following intramuscular injection of an equivalentconcentration or dose of the active agent to the subject. In certainembodiments, the pharmaceutical composition following mucosaladministration yields a peak concentration (C_(max)) of the biologicallyactive agent in the blood plasma or CNS of the subject that is about 50%or greater than the peak concentration of the biologically active agentin the blood plasma or CNS following intramuscular injection of anequivalent concentration or dose of the active agent.

In alternate embodiments of the invention, the pharmaceuticalcompositions comprising a permeabilizing agent and a biologically activeagent are effective following mucosal administration to yield enhancedbioavailability by yielding an area under concentration curve (AUC) ofthe biologically active agent in a blood plasma CNS tissue or fluid ofthe subject that is about 25% or greater compared to an AUC of thebiologically active agent in a blood plasma CNS tissue or fluidfollowing intramuscular injection of an equivalent concentration or doseof the active agent to the subject. In certain embodiments, thepharmaceutical compositions yield an area under concentration curve(AUC) of the biologically active agent in a blood plasma or CNS tissueor fluid of the subject that is about 50% or greater compared to an AUCof the biologically active agent in a blood plasma or CNS tissue orfluid following intramuscular injection of an equivalent concentrationor dose of the active agent to the subject.

In additional embodiments of the invention, the pharmaceuticalcompositions comprising a permeabilizing agent and a biologically activeagent are effective following mucosal administration to yield enhancedbioavailability by yielding a time to maximal plasma concentration(t_(max)) of the biologically active agent in a blood plasma or CNStissue or fluid of the subject between about 0.1 to 1.0 hours. Incertain embodiments, the compositions yield a time to maximal plasmaconcentration (t_(max)) of the biologically active agent in a bloodplasma or CNS tissue or fluid of the subject between about 0.2 to 0.5hours.

In other embodiments of the invention, the pharmaceutical compositionscomprising a permeabilizing agent and a biologically active agent areeffective following mucosal administration to yield enhancedbioavailability of the active agent in the CNS, for example by yieldinga peak concentration of the biologically active agent in a CNS tissue orfluid of the subject that is 10% or greater compared to a peakconcentration of the biologically active agent in a blood plasma of thesubject (e.g., wherein the CNS and plasma concentration is measuredcontemporaneously in the same subject following the mucosaladministration). In certain embodiments, compositions of the inventionyield a peak concentration of the biologically active agent in a CNStissue or fluid of the subject that is 20%, 40%, or greater compared toa peak concentration of the active agent in a blood plasma of thesubject.

The methods of the invention for treating or preventing a disease orcondition in a mammalian subject amenable to treatment by therapeuticadministration of one or more of the biologically active agentsidentified herein generally comprise coordinately, mucosallyadministering to the subject a pharmaceutical formulation comprising abiologically active agent (e.g., peptide YY) and an effective amount ofa permeabilizing agent (e.g., a permeabilizing peptide), as describedabove, to enhance mucosal delivery of the biologically active agent.Coordinate administration of the permeabilizing agent reversiblyenhances mucosal epithelial paracellular transport by modulatingepithelial junctional structure and/or physiology in a target mucosalepithelium of the subject. Typically, the permeabilizing agenteffectively inhibits homotypic or heterotypic binding of an epithelialmembrane adhesive protein selected from a junctional adhesion molecule(JAM), occludin, or claudin. In certain embodiments, the step(s) ofcoordinate mucosal administration involves delivery of thepermeabilizing agent before, after, or simultaneous with (e.g., in acombinatorial formulation) delivery of the biologically active agent toa mucosal surface of the subject. In more detailed embodiments, thepermeabilizing agent is coordinately administered with the biologicallyactive agent to a nasal mucosal surface of the subject, for example in acombinatorial or separate nasal spray, gel or powder formulation(s). Inexemplary embodiments, the permeabilizing agent is a permeabilizingpeptide administered coordinately with the biologically active agent toyield enhanced mucosal epithelial paracellular transport of thebiologically active agent. In certain exemplary embodiments, thepermeabilizing peptide comprises from about 4-25, or about 6-15,contiguous amino acids of an extracellular domain of a mammalian JAM,occludin or claudin protein as described above, or a comparable lengthpeptide that exhibits at least 85% amino acid identity with acorresponding reference sequence of an extracellular domain of amammalian JAM, occludin or claudin protein.

In related aspects of the invention, coordinate administration of thepermeabilizing agent and biologically active agent yields a peakconcentration (C_(max)) of the biologically active agent in a bloodplasma or CNS tissue or fluid of the subject that is 25% or greater ascompared to a peak concentration of the biologically active agentfollowing intramuscular injection of an equivalent concentration or doseof the active agent to the subject. In additional embodiments,coordinate administration of the permeabilizing agent and biologicallyactive agent yields an area under concentration curve (AUC) of thebiologically active agent in a blood plasma or CNS tissue or fluid ofthe subject that is 25% or greater compared to an AUC of thebiologically active agent in a blood plasma or CNS tissue or fluidfollowing intramuscular injection of an equivalent concentration or doseof the active agent to the subject. In other embodiments, coordinateadministration of the permeabilizing agent and biologically active agentyields a time to maximal plasma concentration (t_(max)) of thebiologically active agent in a blood plasma or CNS tissue or fluid ofthe subject between 0.2 to 0.5 hours. In still other embodiments,coordinate administration of the permeabilizing agent and biologicallyactive agent yields a peak concentration of the biologically activeagent in a central nervous system (CNS) tissue or fluid of the subjectthat is 10% or greater compared to a peak concentration of thebiologically active agent in a blood plasma of the subject.

In yet additional detailed embodiments, the invention providespermeabilizing peptides and peptide analogs and mimetics for enhancingmucosal epithelial paracellular transport. The subject peptides andpeptide analogs and mimetics typically work within the compositions andmethods of the invention by modulating epithelial junctional structureand/or physiology in a mammalian subject. In certain embodiments, thepeptides and peptide analogs and mimetics effectively inhibit homotypicand/or heterotypic binding of an epithelial membrane adhesive proteinselected from a junctional adhesion molecule (JAM), occludin, orclaudin. In more detailed embodiments, the permeabilizing peptide orpeptide analog comprises from about 4-25 contiguous amino acids of awild-type sequence of an extracellular domain of a mammalian JAM-1,JAM-2, JAM-3, occludin or claudin protein, or an amino acid sequencethat exhibits at least 85% amino acid identity with a correspondingreference sequence of about 4-25 contiguous amino acids of a wild-typesequence of an extracellular domain of a mammalian JAM-1, JAM-2, JAM-3,occludin or claudin protein. In exemplary embodiments, thepermeabilizing peptide or peptide analog is a human JAM peptide (e.g.,human JAM-1) having a wild-type amino acid sequence or exhibiting one ormore amino acid mutations in comparison to a corresponding wild-typesequence of the same human JAM protein, wherein the mutation(s)correspond to a structural feature identified in a different human JAMprotein or a homologous JAM protein found in a different species.

In more detailed embodiments, (see, e.g., U.S. patent applicationentitled COMPOSITIONS AND METHODS FOR MODULATING PHYSIOLOGY OFEPITHELIAL JUNCTIONAL ADHESION MOLECULES FOR ENHANCED MUCOSAL DELIVERYOF THERAPEUTIC COMPOUNDS, filed by Quay on Jun. 28, 2002, incorporatedherein by reference) the permeabilizing peptide is between about 4-25amino acids in length, and includes one or more contiguous sequenceelements selected from: V R (I, V, A) P, (SEQ ID NO: 1); (V, A, I) K L(S, T) C A Y, (SEQ ID NO: 2); or E D (T, S) G T Y (T,R) C (M, E), (SEQID NO: 3). In one such embodiment, the peptide will include aconservative sequence motif V R (I, V, A) P, (SEQ ID NO: 1), wherein thethird position of the motif may be represented by one of the alternativeamino acid residues I, V, or A. In another such embodiment, the peptidewill include a conservative sequence motif (V, A, I) K L (S, T) C A Y,(SEQ ID NO: 2), wherein the first position of the motif may berepresented by one of the alternative amino acid residues V, A, or I,and the fourth position of the motif may be represented by one of thealternative amino acid residues S or T. In yet another such embodiment,the peptide will include a conservative sequence motif E D (T, S) G T Y(T,R) C (M, E), (SEQ ID NO: 3), wherein the third position of the motifmay be represented by one of the alternative amino acid residues T or S,the seventh position of the motif may be represented by one of thealternative amino acid residues T or R, and the ninth position of themotif may be represented by one of the alternative residues M or E. Inexemplary embodiments, the permeabilizing peptide is between about 4-25amino acids in length and includes one or more contiguous sequenceelements selected from wild-type human JAM-1 peptide sequences VRIP,(SEQ ID NO: 4), VKLSCAY, (SEQ ID NO: 5), and/or EDTGTYTCM, (SEQ ID NO:9).

Candidate permeabilizing peptides of human JAM-1 include, but are notlimited to, SVTVHSSEPE, (SEQ ID NO: 11), VRIPENNPVK, (SEQ ID NO: 12),LSCAYSGFSS, (SEQ ID NO: 13), PRVEWKFDQG, (SEQ ID NO: 14), DTTRLVCYNN,(SEQ ID NO: 15), KITASYEDRV, (SEQ ID NO: 16), TFLPTGITFK, (SEQ ID NO:17), SVTREDTGTY, (SEQ ID NO: 18), TCMVSEEGGN, (SEQ ID NO: 19),SYGEVKVKLI, (SEQ ID NO: 20), VLVPPSKPTV, (SEQ ID NO: 21), NIPSSATIGN,(SEQ ID NO: 22), RAVLTCSEQD, (SEQ ID NO: 23), GSPPSEYTWF, (SEQ ID NO:24), KDGIVMPTNP, (SEQ ID NO: 25), KSTRAFSNSS, (SEQ ID NO: 26),YVLNPTTGEL, (SEQ ID NO: 27), VFDPLSASDT, (SEQ ID NO: 28), GEYSCEARNG,(SEQ ID NO: 29), YGTPMTSNAV, (SEQ ID NO: 30), RMEAVERNVG, (SEQ ID NO:31). Human JAM-1 peptides further include, SVTVH, (SEQ ID NO: 32),SSEPEVRIPE, (SEQ ID NO: 33), NNPVKLSCAY, (SEQ ID NO: 34), SGFSSPRVEW,(SEQ ID NO: 35), KFDQGDTTRL, (SEQ ID NO: 36), VCYNNKITAS, (SEQ ID NO:37), YEDRVTFLPT, (SEQ ID NO: 38), GITFKSVTRE, (SEQ ID NO: 39),DTGTYTCMVS, (SEQ ID NO: 40), EEGGNSYGEV, (SEQ ID NO: 41), KVKLIVLVPP,(SEQ ID NO: 42), SKPTVNIPSS, (SEQ ID NO: 43), ATIGNRAVLT, (SEQ ID NO:44), CSEQDGSPPS, (SEQ ID NO: 45), EYTWFKDGIV, (SEQ ID NO: 46),MPTNPKSTRA, (SEQ ID NO: 47), FSNSSYVLNP, (SEQ ID NO: 48), TTGELVFDPL,(SEQ ID NO: 49), SASDTGEYSC, (SEQ ID NO: 50), EARNGYGTPM, (SEQ ID NO:51), TSNAVRMEAV, (SEQ ID NO: 52), ERNVGVI, (SEQ ID NO: 53). Human JAM-1peptides further include, SVTVHSSE, (SEQ ID NO: 54), PEVRIPEN, (SEQ IDNO: 55), NPVKLSCA, (SEQ ID NO: 56), YSGFSSPR, (SEQ ID NO: 57), VEWKFDQG,(SEQ ID NO: 58), DTTRLVCY, (SEQ ID NO: 59), NNKITASY, (SEQ ID NO: 60),EDRVTFLP, (SEQ ID NO: 61), TGITFKSV, (SEQ ID NO: 62), TREDTGTY, (SEQ IDNO: 63), TCMVSEEG, (SEQ ID NO: 64), GNSYGEVK, (SEQ ID NO: 65), VKLIVLVP,(SEQ ID NO: 66), PSKPTVNI, (SEQ ID NO: 67), PSSATIGN, (SEQ ID NO: 68),RAVLTCSE, (SEQ ID NO: 69), QDGSPPSE, (SEQ ID NO: 70), YTWFKDGI, (SEQ IDNO: 71), VMPTNPKS, (SEQ ID NO: 72), TRAFSNSS, (SEQ ID NO: 73), YVLNPTTG,(SEQ ID NO: 74), ELVFDPLS, (SEQ ID NO: 75), ASDTGEYS, (SEQ ID NO: 76),CEARNGYG, (SEQ ID NO: 77), TPMTSNAV, (SEQ ID NO: 78), RMEAVERN, (SEQ IDNO: 79), VGVI, (SEQ ID NO: 80). Human JAM-1 peptides further include,SVTV, (SEQ ID NO: 81), HSSEPEVR, (SEQ ID NO: 82), IPENNPVK, (SEQ ID NO:83), LSCAYSGF, (SEQ ID NO: 84), SSPRVEWK, (SEQ ID NO: 85), FDQGDTTR,(SEQ ID NO: 86), LVCYNNKI, (SEQ ID NO: 87), TASYEDRV, (SEQ ID NO: 88),TFLPTGIT, (SEQ ID NO: 89), FKSVTRED, (SEQ ID NO: 90), TGTYTCMV, (SEQ IDNO: 91), SEEGGNSY, (SEQ ID NO: 92), GEVKVKLI, (SEQ ID NO: 93), VLVPPSKP,(SEQ ID NO: 94), TVNIPSSA, (SEQ ID NO: 95), TIGNRAVL, (SEQ ID NO: 96),TCSEQDGS, (SEQ ID NO: 97), PPSEYTWF, (SEQ ID NO: 98), KDGIVMPT, (SEQ IDNO: 99), NPKSTRAF, (SEQ ID NO: 100), SNSSYVLN, (SEQ ID NO: 101),PTTGELVF, (SEQ ID NO: 102), DPLSASDT, (SEQ ID NO: 103), GEYSCEAR, (SEQID NO: 104), NGYGTPMT, (SEQ ID NO: 105), SNAVRMEA, (SEQ ID NO: 106),VERNVGVI, (SEQ ID NO: 107).

Exemplary permeabilizing peptides of human JAM-1 include but are notlimited to VR(I,V,A)P, (SEQ ID NO: 1), VR(I)P, (SEQ ID NO: 4), PVR(I)PE,(SEQ ID NO: 108), PEVR(I)PEN, (SEQ ID NO: 55), EPEVR(I)PENN, (SEQ ID NO:109), SEPEVR(I)PENNP, (SEQ ID NO: 110), SSEPEVR(I)PENNPV, (SEQ ID NO:111), HSSEPEVR(I)PENNPVK, (SEQ ID NO: 112), VHSSEPEVR(I)PENNPVKL, (SEQID NO: 113), TVHSSEPEVR(I)PENNPVKLS, (SEQ ID NO: 114), VR(I)PE, (SEQ IDNO: 115), VR(I)PEN, (SEQ ID NO: 116), VR(I)PENN, (SEQ ID NO: 117),VR(I)PENNP, (SEQ ID NO: 118), VR(I)PENNPV, (SEQ ID NO: 119),VR(I)PENNPVK, (SEQ ID NO: 120), VR(I)PENNPVKL, (SEQ ID NO: 121),VR(I)PENNPVKLS, (SEQ ID NO: 122), EVR(I)P, (SEQ ID NO: 123), PEVR(I)P,(SEQ ID NO: 124), EPEVR(I)P, (SEQ ID NO: 125), SEPEVR(I)P, (SEQ ID NO:126), SSEPEVR(I)P, (SEQ ID NO: 127), HSSEPEVR(I)P, (SEQ ID NO: 128),VHSSEPEVR(I)P, (SEQ ID NO: 129), TVHSSEPEVR(I)P, (SEQ ID NO: 130).

Exemplary permeabilizing human JAM-1 peptides further include, VR(V)P,(SEQ ID NO: 131), PVR(V)PE, (SEQ ID NO: 132), PEVR(V)PEN, (SEQ ID NO:133), EPEVR(V)PENN, (SEQ ID NO: 134), SEPEVR(V)PENNP, (SEQ ID NO: 135),SSEPEVR(V)PENNPV, (SEQ ID NO: 136), HSSEPEVR(V)PENNPVK, (SEQ ID NO:137), VHSSEPEVR(V)PENNPVKL, (SEQ ID NO: 138), TVHSSEPEVR(V)PENNPVKLS,(SEQ ID NO: 139), VR(V)PE, (SEQ ID NO: 140), VR(V)PEN, (SEQ ID NO: 141),VR(V)PENN, (SEQ ID NO: 142), VR(V)PENNP, (SEQ ID NO: 143), VR(V)PENNPV,(SEQ ID NO: 144), VR(V)PENNPVK, (SEQ ID NO: 145), VR(V)PENNPVKL, (SEQ IDNO: 146), VR(V)PENNPVKLS, (SEQ ID NO: 147), EVR(V)P, (SEQ ID NO: 148),PEVR(V)P, (SEQ ID NO: 149), EPEVR(V)P, (SEQ ID NO: 150), SEPEVR(V)P,(SEQ ID NO: 151), SSEPEVR(V)P, (SEQ ID NO: 152), HSSEPEVR(V)P, (SEQ IDNO: 153), VHSSEPEVR(V)P, (SEQ ID NO: 154), TVHSSEPEVR(V)P, (SEQ ID NO:155), VR(A)P, (SEQ ID NO: 156), PVR(A)PE, (SEQ ID NO: 157), PEVR(A)PEN,(SEQ ID NO: 158), EPEVR(A)PENN, (SEQ ID NO: 159), SEPEVR(A)PENNP, (SEQID NO: 160), SSEPEVR(A)PENNPV, (SEQ ID NO: 161), HSSEPEVR(A)PENNPVK,(SEQ ID NO: 162), VHSSEPEVR(A)PENNPVKL, (SEQ ID NO: 163),TVHSSEPEVR(A)PENNPVKLS, (SEQ ID NO: 164), VR(A)PE, (SEQ ID NO: 165),VR(A)PEN, (SEQ ID NO: 166), VR(A)PENN, (SEQ ID NO: 167), VR(A)PENNP,(SEQ ID NO: 168), VR(A)PENNPV, (SEQ ID NO: 169), VR(A)PENNPVK, (SEQ IDNO: 170), VR(A)PENNPVKL, (SEQ ID NO: 171), VR(A)PENNPVKLS, (SEQ ID NO:172), EVR(A)P, (SEQ ID NO: 173), PEVR(A)P, (SEQ ID NO: 174), EPEVR(A)P,(SEQ ID NO: 175), SEPEVR(A)P, (SEQ ID NO: 176), SSEPEVR(A)P, (SEQ ID NO:177), HSSEPEVR(A)P, (SEQ ID NO: 178), VHSSEPEVR(A)P, (SEQ ID NO: 179),TVHSSEPEVR(A)P, (SEQ ID NO: 180).

Exemplary permeabilizing human JAM-1 peptides further include,(V,A,I)KL(S,T)CAY, (SEQ ID NO: 2), (V)KL(S)CAY, (SEQ ID NO: 5),P(V)KL(S)CAYS, (SEQ ID NO: 181), NP(V)KL(S)CAYSG, (SEQ ID NO: 182),NNP(V)KL(S)CAYSGF, (SEQ ID NO: 183), ENNP(V)KL(S)CAYSGFS, (SEQ ID NO:184), PENNP(V)KL(S)CAYSGFSS, (SEQ ID NO: 185), IPENNP(V)KL(S)CAYSGFSSP,(SEQ ID NO: 186), RIPENNP(V)KL(S)CAYSGFSSPR, (SEQ ID NO: 187),P(V)KL(S)CAY, (SEQ ID NO: 188), NP(V)KL(S)CAY, (SEQ ID NO: 189),NNP(V)KL(S)CAY, (SEQ ID NO: 190), ENNP(V)KL(S)CAY, (SEQ ID NO: 191),PENNP(V)KL(S)CAY, (SEQ ID NO: 192), IPENNP(V)KL(S)CAY, (SEQ ID NO: 193),RIPENNP(V)KL(S)CAY, (SEQ ID NO: 194), (V)KL(S)CAYS, (SEQ ID NO: 195),(V)KL(S)CAYSG, (SEQ ID NO: 196), (V)KL(S)CAYSGF, (SEQ ID NO: 197),(V)KL(S)CAYSGFS, (SEQ ID NO: 198), (V)KL(S)CAYSGFSS, (SEQ ID NO: 199),(V)KL(S)CAYSGFSSP, (SEQ ID NO: 200), (V)KL(S)CAYSGFSSPR, (SEQ ID NO:201), (V)KL(T)CAY, (SEQ ID NO: 202), (V)KL(T)CAY, (SEQ ID NO: 203),P(V)KL(T)CAYS, (SEQ ID NO: 204), NP(V)KL(T)CAYSG, (SEQ ID NO: 205),NNP(V)KL(T)CAYSGF, (SEQ ID NO: 206), ENNP(V)KL(T)CAYSGFS, (SEQ ID NO:207), PENNP(V)KL(T)CAYSGFSS, (SEQ ID NO: 208), IPENNP(V)KL(T)CAYSGFSSP,(SEQ ID NO: 209), RIPENNP(V)KL(T)CAYSGFSSPR, (SEQ ID NO: 210),P(V)KL(T)CAY, (SEQ ID NO: 211), NP(V)KL(T)CAY, (SEQ ID NO: 212),NNP(V)KL(T)CAY, (SEQ ID NO: 213), ENNP(V)KL(T)CAY, (SEQ ID NO: 214),PENNP(V)KL(T)CAY, (SEQ ID NO: 215), IPENNP(V)KL(T)CAY, (SEQ ID NO: 216),RIPENNP(V)KL(T)CAY, (SEQ ID NO: 217), (V)KL(T)CAYS, (SEQ ID NO: 218),(V)KL(T)CAYSG, (SEQ ID NO: 219), (V)KL(T)CAYSGF, (SEQ ID NO: 220),(V)KL(T)CAYSGFS, (SEQ ID NO: 221), (V)KL(T)CAYSGFSS, (SEQ ID NO: 222),(V)KL(T)CAYSGFSSP, (SEQ ID NO: 223), (V)KL(T)CAYSGFSSPR, (SEQ ID NO:224).

Exemplary permeabilizing human JAM-1 peptides further include,(A)KL(S)CAY, (SEQ ID NO: 225), (A)KL(S)CAY, (SEQ ID NO: 226),P(A)KL(S)CAYS, (SEQ ID NO: 227), NP(A)KL(S)CAYSG, (SEQ ID NO: 228),NNP(A)KL(S)CAYSGF, (SEQ ID NO: 229), ENNP(A)KL(S)CAYSGFS, (SEQ ID NO:230), PENNP(A)KL(S)CAYSGFSS, (SEQ ID NO: 231), IPENNP(A)KL(S)CAYSGFSSP,(SEQ ID NO: 232), RIPENNP(A)KL(S)CAYSGFSSPR, (SEQ ID NO: 233),P(A)KL(S)CAY, (SEQ ID NO: 234), NP(A)KL(S)CAY, (SEQ ID NO: 235),NNP(A)KL(S)CAY, (SEQ ID NO: 236), ENNP(A)KL(S)CAY, (SEQ ID NO: 237),PENNP(A)KL(S)CAY, (SEQ ID NO: 238), IPENNP(A)KL(S)CAY, (SEQ ID NO: 239),RIPENNP(A)KL(S)CAY, (SEQ ID NO: 240), (A)KL(S)CAYS, (SEQ ID NO: 241),(A)KL(S)CAYSG, (SEQ ID NO: 242), (A)KL(S)CAYSGF, (SEQ ID NO: 243),(A)KL(S)CAYSGFS, (SEQ ID NO: 244), (A)KL(S)CAYSGFSS, (SEQ ID NO: 245),(A)KL(S)CAYSGFSSP, (SEQ ID NO: 246), (A)KL(S)CAYSGFSSPR, (SEQ ID NO:247), (A)KL(T)CAY, (SEQ ID NO: 248), (A)KL(T)CAY, (SEQ ID NO: 249),P(A)KL(T)CAYS, (SEQ ID NO: 250), NP(A)KL(T)CAYSG, (SEQ ID NO: 251),NNP(A)KL(T)CAYSGF, (SEQ ID NO: 252), ENNP(A)KL(T)CAYSGFS, (SEQ ID NO:253), PENNP(A)KL(T)CAYSGFSS, (SEQ ID NO: 254), IPENNP(A)KL(T)CAYSGFSSP,(SEQ ID NO: 255), RIPENNP(A)KL(T)CAYSGFSSPR, (SEQ ID NO: 256),P(A)KL(T)CAY, (SEQ ID NO: 257), NP(A)KL(T)CAY, (SEQ ID NO: 258),NNP(A)KL(T)CAY, (SEQ ID NO: 259), ENNP(A)KL(T)CAY, (SEQ ID NO: 260),PENNP(A)KL(T)CAY, (SEQ ID NO: 261), IPENNP(A)KL(T)CAY, (SEQ ID NO: 262),RIPENNP(A)KL(T)CAY, (SEQ ID NO: 263), (A)KL(T)CAYS, (SEQ ID NO: 264),(A)KL(T)CAYSG, (SEQ ID NO: 265), (A)KL(T)CAYSGF, (SEQ ID NO: 266),(A)KL(T)CAYSGFS, (SEQ ID NO: 267), (A)KL(T)CAYSGFSS, (SEQ ID NO: 268),(A)KL(T)CAYSGFSSP, (SEQ ID NO: 269), (A)KL(T)CAYSGFSSPR, (SEQ ID NO:270).

Exemplary permeabilizing human JAM-1 peptides further include,ED(T,S)GTY(T,R)C(M,E), (SEQ ID NO: 3), ED(T)GTY(T)C(M), (SEQ ID NO: 9),RED(T)GTY(T)C(M)V, (SEQ ID NO: 271), TRED(T)GTY(T)C(M)VS, (SEQ ID NO:272), VTRED(T)GTY(T)C(M)VSE, (SEQ ID NO: 273), SVTRED(T)GTY(T)C(M)VSEE,(SEQ ID NO: 274), KSVTRED(T)GTY(T)C(M)VSEEG, (SEQ ID NO: 275),RED(T)GTY(T)C(M), (SEQ ID NO: 276), TRED(T)GTY(T)C(M), (SEQ ID NO: 277),VTRED(T)GTY(T)C(M), (SEQ ID NO: 278), SVTRED(T)GTY(T)C(M), (SEQ ID NO:279), KSVTRED(T)GTY(T)C(M), (SEQ ID NO: 280), ED(T)GTY(T)C(M)V, (SEQ IDNO: 281), ED(T)GTY(T)C(M)VS, (SEQ ID NO: 282), ED(T)GTY(T)C(M)VSE, (SEQID NO: 283), ED(T)GTY(T)C(M)VSEE, (SEQ ID NO: 284),ED(T)GTY(T)C(M)VSEEG, (SEQ ID NO: 285), ED(T)GTY(T)C(E), (SEQ ID NO:286), RED(T)GTY(T)C(E)V, (SEQ ID NO: 287), TRED(T)GTY(T)C(E)VS, (SEQ IDNO: 288), VTRED(T)GTY(T)C(E)VSE, (SEQ ID NO: 289),SVTRED(T)GTY(T)C(E)VSEE, (SEQ ID NO: 290), KSVTRED(T)GTY(T)C(E)VSEEG,(SEQ ID NO: 291), RED(T)GTY(T)C(E), (SEQ ID NO: 292), TRED(T)GTY(T)C(E),(SEQ ID NO: 293), VTRED(T)GTY(T)C(E), (SEQ ID NO: 294),SVTRED(T)GTY(T)C(E), (SEQ ID NO: 295), KSVTRED(T)GTY(T)C(E), (SEQ ID NO:296), ED(T)GTY(T)C(E)V, (SEQ ID NO: 297), ED(T)GTY(T)C(E)VS, (SEQ ID NO:298), ED(T)GTY(T)C(E)VSE, (SEQ ID NO: 299), ED(T)GTY(T)C(E)VSEE, (SEQ IDNO: 300), ED(T)GTY(T)C(E)VSEEG, (SEQ ID NO: 301), ED(T)GTY(R)C(M), (SEQID NO: 302), RED(T)GTY(T)C(M)V, (SEQ ID NO: 303), TRED(T)GTY(T)C(M)VS,(SEQ ID NO: 304), VTRED(T)GTY(T)C(M)VSE, (SEQ ID NO: 305),SVTRED(T)GTY(T)C(M)VSEE, (SEQ ID NO: 306), KSVTRED(T)GTY(T)C(M)VSEEG,(SEQ ID NO: 307), RED(T)GTY(T)C(M), (SEQ ID NO: 308), TRED(T)GTY(T)C(M),(SEQ ID NO: 309), VTRED(T)GTY(T)C(M), (SEQ ID NO: 310),SVTRED(T)GTY(T)C(M), (SEQ ID NO: 311), KSVTRED(T)GTY(T)C(M), (SEQ ID NO:312), ED(T)GTY(T)C(M)V, (SEQ ID NO: 313), ED(T)GTY(T)C(M)VS, (SEQ ID NO:314), ED(T)GTY(T)C(M)VSE, (SEQ ID NO: 315), ED(T)GTY(T)C(M)VSEE, (SEQ IDNO: 316), ED(T)GTY(T)C(M)VSEEG, (SEQ ID NO: 317).

Exemplary permeabilizing human JAM-1 peptides further include,ED(T)GTY(R)C(E), (SEQ ID NO: 318), RED(T)GTY(T)C(M)V, (SEQ ID NO: 319),TRED(T)GTY(T)C(M)VS, (SEQ ID NO: 320), VTRED(T)GTY(T)C(M)VSE, (SEQ IDNO: 321), SVTRED(T)GTY(T)C(M)VSEE, (SEQ ID NO: 322),KSVTRED(T)GTY(T)C(M)VSEEG, (SEQ ID NO: 323), RED(T)GTY(T)C(M), (SEQ IDNO: 324), TRED(T)GTY(T)C(M), (SEQ ID NO: 325), VTRED(T)GTY(T)C(M), (SEQID NO: 326), SVTRED(T)GTY(T)C(M), (SEQ ID NO: 327),KSVTRED(T)GTY(T)C(M), (SEQ ID NO: 328), ED(T)GTY(T)C(M)V, (SEQ ID NO:329), ED(T)GTY(T)C(M)VS, (SEQ ID NO: 330), ED(T)GTY(T)C(M)VSE, (SEQ IDNO: 331), ED(T)GTY(T)C(M)VSEE, (SEQ ID NO: 332), ED(T)GTY(T)C(M)VSEEG,(SEQ ID NO: 333), ED(S)GTY(T)C(M), (SEQ ID NO: 334), RED(T)GTY(T)C(M)V,(SEQ ID NO: 335), TRED(T)GTY(T)C(M)VS, (SEQ ID NO: 336),VTRED(T)GTY(T)C(M)VSE, (SEQ ID NO: 337), SVTRED(T)GTY(T)C(M)VSEE, (SEQID NO: 338), KSVTRED(T)GTY(T)C(M)VSEEG, (SEQ ID NO: 339),RED(T)GTY(T)C(M), (SEQ ID NO: 340), TRED(T)GTY(T)C(M), (SEQ ID NO: 341),VTRED(T)GTY(T)C(M), (SEQ ID NO: 342), SVTRED(T)GTY(T)C(M), (SEQ ID NO:343), KSVTRED(T)GTY(T)C(M), (SEQ ID NO: 344), ED(T)GTY(T)C(M)V, (SEQ IDNO: 345), ED(T)GTY(T)C(M)VS, (SEQ ID NO: 346), ED(T)GTY(T)C(M)VSE, (SEQID NO: 347), ED(T)GTY(T)C(M)VSEE, (SEQ ID NO: 348),ED(T)GTY(T)C(M)VSEEG, (SEQ ID NO: 349), ED(S)GTY(T)C(E), (SEQ ID NO:350), RED(S)GTY(T)C(E)V, (SEQ ID NO: 351), TRED(S)GTY(T)C(E)VS, (SEQ IDNO: 352), VTRED(S)GTY(T)C(E)VSE, (SEQ ID NO: 353),SVTRED(S)GTY(T)C(E)VSEE, (SEQ ID NO: 354), KSVTRED(S)GTY(T)C(E)VSEEG,(SEQ ID NO: 355), RED(S)GTY(T)C(E), (SEQ ID NO: 356), TRED(S)GTY(T)C(E),(SEQ ID NO: 357), VTRED(S)GTY(T)C(E), (SEQ ID NO: 358),SVTRED(S)GTY(T)C(E), (SEQ ID NO: 359), KSVTRED(S)GTY(T)C(E), (SEQ ID NO:360), ED(S)GTY(T)C(E)V, (SEQ ID NO: 361), ED(S)GTY(T)C(E)VS, (SEQ ID NO:362), ED(S)GTY(T)C(E)VSE, (SEQ ID NO: 363), ED(S)GTY(T)C(E)VSEE, (SEQ IDNO: 364), ED(S)GTY(T)C(E)VSEEG, (SEQ ID NO: 365).

Exemplary permeabilizing human JAM-1 peptides further include,ED(S)GTY(R)C(M), (SEQ ID NO: 366), RED(S)GTY(R)C(M)V, (SEQ ID NO: 367),TRED(S)GTY(R)C(M)VS, (SEQ ID NO: 368), VTRED(S)GTY(R)C(M)VSE, (SEQ IDNO: 369), SVTRED(S)GTY(R)C(M)VSEE, (SEQ ID NO: 370),KSVTRED(S)GTY(R)C(M)VSEEG, (SEQ ID NO: 371), RED(S)GTY(R)C(M), (SEQ IDNO: 372), TRED(S)GTY(R)C(M), (SEQ ID NO: 373), VTRED(S)GTY(R)C(M), (SEQID NO: 374), SVTRED(S)GTY(R)C(M), (SEQ ID NO: 375),KSVTRED(S)GTY(R)C(M), (SEQ ID NO: 376), ED(S)GTY(R)C(M)V, (SEQ ID NO:377), ED(S)GTY(R)C(M)VS, (SEQ ID NO: 378), ED(S)GTY(R)C(M)VSE, (SEQ IDNO: 379), ED(S)GTY(R)C(M)VSEE, (SEQ ID NO: 380), ED(S)GTY(R)C(M)VSEEG,(SEQ ID NO: 381).

Exemplary permeabilizing human JAM-1 peptides further include,ED(S)GTY(R)C(E), (SEQ ID NO: 382), RED(S)GTY(R)C(E)V, (SEQ ID NO: 383),TRED(S)GTY(R)C(E)VS, (SEQ ID NO: 384), VTRED(S)GTY(R)C(E)VSE, (SEQ IDNO: 385), SVTRED(S)GTY(R)C(E)VSEE, (SEQ ID NO: 386),KSVTRED(S)GTY(R)C(E)VSEEG, (SEQ ID NO: 387), RED(S)GTY(R)C(E), (SEQ IDNO: 388), TRED(S)GTY(R)C(E), (SEQ ID NO: 389), VTRED(S)GTY(R)C(E), (SEQID NO: 390), SVTRED(S)GTY(R)C(E), (SEQ ID NO: 391),KSVTRED(S)GTY(R)C(E), (SEQ ID NO: 392), ED(S)GTY(R)C(E)V, (SEQ ID NO:393), ED(S)GTY(R)C(E)VS, (SEQ ID NO: 394), ED(S)GTY(R)C(E)VSE, (SEQ IDNO: 395), ED(S)GTY(R)C(E)VSEE, (SEQ ID NO: 396), ED(S)GTY(R)C(E)VSEEG,(SEQ ID NO: 397).

Candidate permeabilizing peptides of human JAM-2 include, but are notlimited to AVNLKSSNRT, (SEQ ID NO: 398), PVVQEFESVE, (SEQ ID NO: 399),LSCIITDSQT, (SEQ ID NO: 400), SDPRIEWKKI, (SEQ ID NO: 401), QDEQTTYVFF,(SEQ ID NO: 402), DNKIQGDLAG, (SEQ ID NO: 403), RAEILGKTSL, (SEQ ID NO:404), KIWNVTRRDS, (SEQ ID NO: 405), ALYRCEVVAR, (SEQ ID NO: 406),NDRKEIDEIV, (SEQ ID NO: 407), IELTVQVKPV, (SEQ ID NO: 408), TPVCRVPKAV,(SEQ ID NO: 409), PVGKMATLHC, (SEQ ID NO: 410), QESEGHPRPH, (SEQ ID NO:411), YSWYRNDVPL, (SEQ ID NO: 412), PTDSRANPRF, (SEQ ID NO: 413),RNSSFHLNSE, (SEQ ID NO: 414), TGTLVFTAVH, (SEQ ID NO: 415), KDDSGQYYCI,(SEQ ID NO: 416), ASNDAGSARC, (SEQ ID NO: 417), EEQEMEVYDLN, (SEQ ID NO:418).

Candidate permeabilizing peptides of human JAM-2 further include AVNLK,(SEQ ID NO: 419), SSNRTPVVQE, (SEQ ID NO: 420), FESVELSCII, (SEQ ID NO:421), TDSQTSDPRI, (SEQ ID NO: 422), EWKKIQDEQT, (SEQ ID NO: 423),TYVFFDNKIQ, (SEQ ID NO: 424), GDLAGRAEIL, (SEQ ID NO: 425), GKTSLKIWNV,(SEQ ID NO: 426), TRRDSALYRC, (SEQ ID NO: 427), EVVARNDRKE, (SEQ ID NO:428), IDEIVIELTV, (SEQ ID NO: 429), QVKPVTPVCR, (SEQ ID NO: 430),VPKAVPVGKM, (SEQ ID NO: 431), ATLHCQESEG, (SEQ ID NO: 432), HPRPHYSWYR,(SEQ ID NO: 433), NDVPLPTDSR, (SEQ ID NO: 434), ANPRFRNSSF, (SEQ ID NO:435), HLNSETGTLV, (SEQ ID NO: 436), FTAVHKDDSG, (SEQ ID NO: 437),QYYCIASNDA, (SEQ ID NO: 438), GSARCEEQEM, (SEQ ID NO: 439), EVYDLN, (SEQID NO: 440).

Candidate permeabilizing peptides of human JAM-2 further includeAVNLKSSN, (SEQ ID NO: 441), RTPVVQEF, (SEQ ID NO: 442), ESVELSCI, (SEQID NO: 443), ITDSQTSD, (SEQ ID NO: 444), PRIEWKKI, (SEQ ID NO: 790),QDEQTTYV, (SEQ ID NO: 445), FFDNKIQG, (SEQ ID NO: 446), DLAGRAEI, (SEQID NO: 447), LGKTSLKI, (SEQ ID NO: 448), WNVTRRDS, (SEQ ID NO: 449),ALYRCEVV, (SEQ ID NO: 450), ARNDRKEI, (SEQ ID NO: 451), DEIVIELT, (SEQID NO: 452), VQVKPVTP, (SEQ ID NO: 453), VCRVPKAV, (SEQ ID NO: 454),PVGKMATL, (SEQ ID NO: 455), HCQESEGH, (SEQ ID NO: 456), PRPHYSWY, (SEQID NO: 457), RNDVPLPT, (SEQ ID NO: 458), DSRANPRF, (SEQ ID NO: 459),RNSSFHLN, (SEQ ID NO: 460), SETGTLVF, (SEQ ID NO: 461), TAVHKDDS, (SEQID NO: 462), GQYYCIAS, (SEQ ID NO: 463), NDAGSARC, (SEQ ID NO: 464),EEQEMEVY, (SEQ ID NO: 465), DLN, (SEQ ID NO: 466).

Candidate permeabilizing peptides of human JAM-2 further include AVNL,(SEQ ID NO: 467), KSSNRTPV, (SEQ ID NO: 468), VQEFESVE, (SEQ ID NO:469), LSCIITDS, (SEQ ID NO: 470), QTSDPRIE, (SEQ ID NO: 471), WKKIQDEQ,(SEQ ID NO: 472), TTYVFFDN, (SEQ ID NO: 473), KIQGDLAG, (SEQ ID NO:474), RAEILGKT, (SEQ ID NO: 475), SLKIWNVT, (SEQ ID NO: 476), RRDSALYR,(SEQ ID NO: 477), CEVVARND, (SEQ ID NO: 478), RKEIDEIV, (SEQ ID NO:479), IELTVQVK, (SEQ ID NO: 480), PVTPVCRV, (SEQ ID NO: 481), PKAVPVGK,(SEQ ID NO: 482), MATLHCQE, (SEQ ID NO: 483), SEGHPRPH, (SEQ ID NO:484), YSWYRNDV, (SEQ ID NO: 485), PLPTDSRA, (SEQ ID NO: 486), NPRFRNSS,(SEQ ID NO: 487), FHLNSETG, (SEQ ID NO: 488), TLVFTAVH, (SEQ ID NO:489), KDDSGQYY, (SEQ ID NO: 490), CIASNDAG, (SEQ ID NO: 491), SARCEEQE,(SEQ ID NO: 492), MEVYDLN, (SEQ ID NO: 493).

Exemplary permeabilizing peptides of human JAM-3 include, but are notlimited to, GFSAPKDQQV, (SEQ ID NO: 494), VTAVEYQEAI, (SEQ ID NO: 495),LACKTPKKTV, (SEQ ID NO: 496), SSRLEWKKLG, (SEQ ID NO: 497), RSVSFVYYQQ,(SEQ ID NO: 498), TLQGDFKNRA, (SEQ ID NO: 499), EMIDFNIRIK, (SEQ ID NO:500), NVTRSDAGKY, (SEQ ID NO: 501), RCEVSAPSEQ, (SEQ ID NO: 502),GQNLEEDTVT, (SEQ ID NO: 503), LEVLVAPAVP, (SEQ ID NO: 504), SCEVPSSALS,(SEQ ID NO: 505), GTVVELRCQD, (SEQ ID NO: 506), KEGNPAPEYT, (SEQ ID NO:507), WFKDGIRLLE, (SEQ ID NO: 508), NPRLGSQSTN, (SEQ ID NO: 509),SSYTMNTKTG, (SEQ ID NO: 510), TLQFNTVSKL, (SEQ ID NO: 511), DTGEYSCEAR,(SEQ ID NO: 512), NSVGYRRCPG, (SEQ ID NO: 513), KRMQVDDLN, (SEQ ID NO:514).

Exemplary permeabilizing peptides of human JAM-3 further include GFSAP,(SEQ ID NO: 515), KDQQVVTAVE, (SEQ ID NO: 516), YQEAILACKT, (SEQ ID NO:517), PKKTVSSRLE, (SEQ ID NO: 518), WKKLGRSVSF, (SEQ ID NO: 519),VYYQQTLQGD, (SEQ ID NO: 520), FKNRAEMIDF, (SEQ ID NO: 521), NIRIKNVTRS,(SEQ ID NO: 522), DAGKYRCEVS, (SEQ ID NO: 523), APSEQGQNLE, (SEQ ID NO:524), EDTVTLEVLV, (SEQ ID NO: 525), APAVPSCEVP, (SEQ ID NO: 526),SSALSGTVVE, (SEQ ID NO: 527), LRCQDKEGNP, (SEQ ID NO: 528), APEYTWFKDG,(SEQ ID NO: 529), IRLLENPRLG, (SEQ ID NO: 530), SQSTNSSYTM, (SEQ ID NO:531), NTKTGTLQFN, (SEQ ID NO: 532), TVSKLDTGEY, (SEQ ID NO: 533),SCEARNSVGY, (SEQ ID NO: 534), RRCPGKRMQV, (SEQ ID NO: 535), DDLN, (SEQID NO: 536).

Exemplary permeabilizing peptides of human JAM-3 further includeGFSAPKDQ, (SEQ ID NO: 537), QVVTAVEY, (SEQ ID NO: 538), QEAILACK, (SEQID NO: 539), TPKKTVSS, (SEQ ID NO: 540), RLEWKKLG, (SEQ ID NO: 541),RSVSFVYY, (SEQ ID NO: 542), QQTLQGDF, (SEQ ID NO: 543), KNRAEMID, (SEQID NO: 544), FNIRIKNV, (SEQ ID NO: 545), TRSDAGKY, (SEQ ID NO: 546),RCEVSAPS, (SEQ ID NO: 547), EQGQNLEE, (SEQ ID NO: 548), DTVTLEVL, (SEQID NO: 549), VAPAVPSC, (SEQ ID NO: 550), EVPSSALS, (SEQ ID NO: 551),GTVVELRC, (SEQ ID NO: 552), QDKEGNPA, (SEQ ID NO: 553), PEYTWFKD, (SEQID NO: 554), GIRLLENP, (SEQ ID NO: 555), RLGSQSTN, (SEQ ID NO: 556),SSYTMNTK, (SEQ ID NO: 557), TGTLQFNT, (SEQ ID NO: 558), VSKLDTGE, (SEQID NO: 559), YSCEARNS, (SEQ ID NO: 560), VGYRRCPG, (SEQ ID NO: 561),KRMQVDDLN, (SEQ ID NO: 562).

Exemplary permeabilizing peptides of human JAM-3 further include GFSA,(SEQ ID NO: 563), PKDQQVVT, (SEQ ID NO: 564), AVEYQEAI, (SEQ ID NO:565), LACKTPKK, (SEQ ID NO: 566), TVSSRLEW, (SEQ ID NO: 567), KKLGRSVS,(SEQ ID NO: 568), FVYYQQTL, (SEQ ID NO: 569), QGDFKNRA, (SEQ ID NO:570), EMIDFNIR, (SEQ ID NO: 571), IKNVTRSD, (SEQ ID NO: 572), AGKYRCEV,(SEQ ID NO: 573), SAPSEQGQ, (SEQ ID NO: 574), NLEEDTVT, (SEQ ID NO:575), LEVLVAPA, (SEQ ID NO: 576), VPSCEVPS, (SEQ ID NO: 577), SALSGTVV,(SEQ ID NO: 578), ELRCQDKE, (SEQ ID NO: 579), GNPAPEYT, (SEQ ID NO:580), WFKDGIRL, (SEQ ID NO: 581), LENPRLGS, (SEQ ID NO: 582), QSTNSSYT,(SEQ ID NO: 583), MNTKTGTL, (SEQ ID NO: 584), QFNTVSKL, (SEQ ID NO:585), DTGEYSCE, (SEQ ID NO: 586), ARNSVGYR, (SEQ ID NO: 587), RCPGKRMQ,(SEQ ID NO: 588), VDDLN, (SEQ ID NO: 589).

Exemplary permeabilizing peptides of human claudin 1 extracellulardomain include, but are not limited to, RIYSYAGDNI, (SEQ ID NO: 590),VTAQAMYEGL, (SEQ ID NO: 591), WMSCVSQSTG, (SEQ ID NO: 592), QIQCKVFDSL,(SEQ ID NO: 593), LNLSSTLQATR, (SEQ ID NO: 594), RIYSY, (SEQ ID NO:595), AGDNIVTAQA, (SEQ ID NO: 596), MYEGLWMSCV, (SEQ ID NO: 597),SQSTGQIQCK, (SEQ ID NO: 598), VFDSLLNLSS, (SEQ ID NO: 599), TLQATR, (SEQID NO: 600), QEFYDPMT, (SEQ ID NO: 601), PVNARYE, (SEQ ID NO: 602),QEFYDPMTPVN, (SEQ ID NO: 603), ARYE, (SEQ ID NO: 604).

Exemplary permeabilizing peptides of human claudin 2 extracellulardomain include, but are not limited to, KTSSYVGASI, (SEQ ID NO: 605),VTAVGFSKGL, (SEQ ID NO: 606), WMECATHSTG, (SEQ ID NO: 607), ITQCDIYSTL,(SEQ ID NO: 608), LGLPADIQAAQ, (SEQ ID NO: 609), KTSSY, (SEQ ID NO:610), VGASIVTAVG, (SEQ ID NO: 611), FSKGLWMECA, (SEQ ID NO: 612),THSTGITQCD, (SEQ ID NO: 613), IYSTLLGLPA, (SEQ ID NO: 614), DIQAAQ, (SEQID NO: 615), RDFYSPL, (SEQ ID NO: 616).

Exemplary permeabilizing peptides of human claudin 3 extracellulardomain include, but are not limited to, RVSAFIGSNI, (SEQ ID NO: 617),ITSQNIWEGL, (SEQ ID NO: 618), WMNCVVQSTG, (SEQ ID NO: 619), QMQCKVYDSL,(SEQ ID NO: 620), LALPQDLQAAR, (SEQ ID NO: 621), RVSAF, (SEQ ID NO:622), IGSNIITSQN, (SEQ ID NO: 623), IWEGLWMNCV, (SEQ ID NO: 624),VQSTGQMQCK, (SEQ ID NO: 625), VYDSLLALPQ, (SEQ ID NO: 626), DLQAAR, (SEQID NO: 627), RDFYNPVV, (SEQ ID NO: 628), PEAQKRE, (SEQ ID NO: 629).

Exemplary permeabilizing peptides of human claudin 4 extracellulardomain include, but are not limited to, RVTAFIGSNI, (SEQ ID NO: 630),VTSQTIWEGL, (SEQ ID NO: 631), WMNCVVQSTG, (SEQ ID NO: 632), QMQCKVYDSL,(SEQ ID NO: 633), LALPQDLQAAR, (SEQ ID NO: 634), RVTAF, (SEQ ID NO:635), IGSNIVTSQT, (SEQ ID NO: 636), IWEGLWMNCV, (SEQ ID NO: 637),VQSTGQMQCK, (SEQ ID NO: 638), VYDSLLALPQ, (SEQ ID NO: 639), DLQAAR, (SEQID NO: 640), QDFYNPLV, (SEQ ID NO: 641), ASGQKRE, (SEQ ID NO: 642).

Exemplary permeabilizing peptides of human claudin 5 extracellulardomain include, but are not limited to, QVTAFLDHNI, (SEQ ID NO: 643),VTAQTTWKGL, (SEQ ID NO: 644), WMSCVVQSTG, (SEQ ID NO: 645), HMQCKVYDSV,(SEQ ID NO: 646), LALSTEVQAAR, (SEQ ID NO: 647), QVTAF, (SEQ ID NO:648), LDHNIVTAQT, (SEQ ID NO: 649), TWKGLWMSCV, (SEQ ID NO: 650),VQSTGHMQCK, (SEQ ID NO: 651), VYDSVLALST, (SEQ ID NO: 652), EVQAAR, (SEQID NO: 653), REFYDPSV, (SEQ ID NO: 654).

Exemplary permeabilizing peptides of human claudin 6 extracellulardomain include, but are not limited to, KVTAFIGNSI, (SEQ ID NO: 655),VVAQVVWEGL, (SEQ ID NO: 656), WMSCVVQSTG, (SEQ ID NO: 657), QMQCKVYDSL,(SEQ ID NO: 658), LALPQDLQAAR, (SEQ ID NO: 659), KVTAF, (SEQ ID NO:660), IGNSIVVAQV, (SEQ ID NO: 661), VWEGLWMSCV, (SEQ ID NO: 662),VQSTGQMQCK, (SEQ ID NO: 663), VYDSLLALPQ, (SEQ ID NO: 664), DLQAAR, (SEQID NO: 665), RDFYNPLV, (SEQ ID NO: 666), AEAQKRE, (SEQ ID NO: 667).

Exemplary permeabilizing peptides of human claudin 7 extracellulardomain include, but are not limited to, QMSSYAGDNI, (SEQ ID NO: 668),ITAQAMYKGL, (SEQ ID NO: 669), WMDCVTQSTG, (SEQ ID NO: 670), MMSCKMYDSV,(SEQ ID NO: 671), LALSAALQATR, (SEQ ID NO: 672), QMSSY, (SEQ ID NO:673), AGDNIITAQA, (SEQ ID NO: 674), MYKGLWMDCV, (SEQ ID NO: 675),TQSTGMMSCK, (SEQ ID NO: 676), MYDSVLALSA, (SEQ ID NO: 677), ALQATR, (SEQID NO: 678), TDFYNPLI, (SEQ ID NO: 679), PTNIKYE, (SEQ ID NO: 680).

Exemplary permeabilizing peptides of human claudin 8 extracellulardomain include, but are not limited to, RVSAFIENNI, (SEQ ID NO: 681),VVFENFWEGL, (SEQ ID NO: 682), WMNCVRQANI, (SEQ ID NO: 683), RMQCKIYDSL,(SEQ ID NO: 684), LALSPDLQAAR, (SEQ ID NO: 685), RVSAF, (SEQ ID NO:686), IENNIVVFEN, (SEQ ID NO: 687), FWEGLWMNCV, (SEQ ID NO: 688),RQANIRMQCK, (SEQ ID NO: 689), IYDSLLALSP, (SEQ ID NO: 690), DLQAAR, (SEQID NO: 691), RDFYNSIV, (SEQ ID NO: 692), NVAQKRE, (SEQ ID NO: 693).

Exemplary permeabilizing peptides of human claudin 9 extracellulardomain include, but are not limited to, KVTAFIGNSI, (SEQ ID NO: 694),VVAQVVWEGL, (SEQ ID NO: 695), WMSCVVQSTG, (SEQ ID NO: 696), QMQCKVYDSL,(SEQ ID NO: 697), LALPQDLQAAR, (SEQ ID NO: 698), KVTAF, (SEQ ID NO:699), IGNSIVVAQV, (SEQ ID NO: 700), VWEGLWMSCV, (SEQ ID NO: 701),VQSTGQMQCK, (SEQ ID NO: 702), VYDSLLALPQ, (SEQ ID NO: 703), DLQAAR, (SEQID NO: 704), QDFYNPLV, (SEQ ID NO: 705), AEALKRE, (SEQ ID NO: 706).

Exemplary permeabilizing peptides of human claudin 10 extracellulardomain include, but are not limited to, KVSTIDGTVI, (SEQ ID NO: 707),TTATYWANLW, (SEQ ID NO: 708), KACVTDSTGV, (SEQ ID NO: 709), SNCKDFPSML,(SEQ ID NO: 710), ALDGYIQACR, (SEQ ID NO: 711), KVSTI, (SEQ ID NO: 712),DGTVITTATY, (SEQ ID NO: 713), WANLWKACVT, (SEQ ID NO: 714), DSTGVSNCKD,(SEQ ID NO: 715), FPSMLALDGY, (SEQ ID NO: 716), IQACR, (SEQ ID NO: 717),EFFDPLF, (SEQ ID NO: 718), VEQKYE, (SEQ ID NO: 719).

Exemplary permeabilizing peptides of human occludin extracellular domaininclude, but are not limited to, DRGYGTSLLG, (SEQ ID NO: 720),GSVGYPYGGS, (SEQ ID NO: 721), GFGSYGSGYG, (SEQ ID NO: 722), YGYGYGYGYG,(SEQ ID NO: 723), GYTDPR, (SEQ ID NO: 724), DRGYG, (SEQ ID NO: 725),TSLLGGSVGY, (SEQ ID NO: 726), PYGGSGFGSY, (SEQ ID NO: 727), GSGYGYGYGY,(SEQ ID NO: 728), GYGYGGYTDPR, (SEQ ID NO: 729), GVNPTAQSSG, (SEQ ID NO:730), SLYGSQIYAL, (SEQ ID NO: 731), CNQFYTPAAT, (SEQ ID NO: 732),GLYVDQYLYH, (SEQ ID NO: 733), YCVVDPQE, (SEQ ID NO: 734), GVNPT, (SEQ IDNO: 735), AQSSGSLYGS, (SEQ ID NO: 736), QIYALCNQFY, (SEQ ID NO: 737),TPAATGLYVD, (SEQ ID NO: 738), QYLYHYCVVD, (SEQ ID NO: 739), PQE, (SEQ IDNO: 740).

Further candidate permeabilizing peptides of human JAM-1 include, butare not limited to, VRIP, (SEQ ID NO: 4), VKLSCAY, (SEQ ID NO: 5),TGITFKSVT, (SEQ ID NO: 6), ITAS, (SEQ ID NO: 7), SVTR, (SEQ ID NO: 8),SVTVHSSEP, (SEQ ID NO: 741), KFDQGDTTR, (SEQ ID NO: 742), EDTGTYTCM,(SEQ ID NO: 9), GEVKVKLIV, (SEQ ID NO: 743), VSEEGGNSY, (SEQ ID NO:744), LVCYNNKIT, (SEQ ID NO: 745), GFSSPRVEW, (SEQ ID NO: 10), VLPPS,(SEQ ID NO: 746), YEDRVTF, (SEQ ID NO: 747), PRVEW, (SEQ ID NO: 748).

Further candidate permeabilizing peptides of human claudin-1 include,but are not limited to, KVFDSLLNLS, (SEQ ID NO: 749), NRIVQEFYDP, (SEQID NO: 750), YAGDNIVTAQ, (SEQ ID NO: 751), VSQSTGQIQC, (SEQ ID NO: 752),MTPVNARYEF, (SEQ ID NO: 753), AMYEGLWMSC, (SEQ ID NO: 754), TTWLGLWMSC,(SEQ ID NO: 755).

Further candidate permeabilizing peptides of human claudin-2 include,but are not limited to, YVGASIVTAV, (SEQ ID NO: 756), GILRDFYSPL, (SEQID NO: 757), VPDSMKFEIG, (SEQ ID NO: 758), DIYSTLLGLP, (SEQ ID NO: 759),GFSLGLWMEC, (SEQ ID NO: 760), ATHSTGITQC, (SEQ ID NO: 761), GFSKGLWMEC,(SEQ ID NO: 762).

Further candidate permeabilizing peptides of human claudin-3 include,but are not limited to, KVYDSLLALP, (SEQ ID NO: 763), NTIIRDFYNP, (SEQID NO: 764), VVPEAQKREM, (SEQ ID NO: 765), NIWEGLWMNC, (SEQ ID NO: 766),VVQSTGQMQC, (SEQ ID NO: 767), FIGSNIITSQ, (SEQ ID NO: 768).

Further candidate permeabilizing peptides of human claudin-4 include,but are not limited to, VASGQKREMG, (SEQ ID NO: 769), NIIQDFYNPL, (SEQID NO: 770), FIGSNIVTSQ, (SEQ ID NO: 771), TIWEGLWMNC, (SEQ ID NO: 772).

Further candidate permeabilizing peptides of human claudin-5 include,but are not limited to, IVVREFYDPS, (SEQ ID NO: 773), VVQSTGHMQC, (SEQID NO: 774), FLDHNIVTAQ, (SEQ ID NO: 775), VPVSQKYELG, (SEQ ID NO: 776),KVYDSVLALS, (SEQ ID NO: 777), TTWKGLWMSC, (SEQ ID NO: 778).

Further candidate permeabilizing peptides of human occludin include, butare not limited to, DRGYGTSLL, (SEQ ID NO: 779), GYGYGYGYG, (SEQ ID NO:780), GSGFGSYGS, (SEQ ID NO: 781), YGYGGYTDP, (SEQ ID NO: 782),GVNPTAQSS, (SEQ ID NO: 783), GSLYGSQIY, (SEQ ID NO: 784), AATGLYVDQ,(SEQ ID NO: 785), ALCNQFYTP, (SEQ ID NO: 786), YLYHYCVVD, (SEQ ID NO:787), GGSVGYPYG, (SEQ ID NO: 788).

In addition to JAM, occludin and claudin peptides, proteins, analogs andmimetics, additional agents for modulating epithelial junctionalphysiology and/or structure are contemplated for use within the methodsand formulations of the invention. Epithelial tight junctions aregenerally impermeable to molecules with radii of approximately 15angstroms, unless treated with junctional physiological control agentsthat stimulate substantial junctional opening as provided within theinstant invention. Among the “secondary” tight junctional regulatorycomponents that will serve as useful targets for secondary physiologicalmodulation within the methods and compositions of the invention, theZO1-ZO2 heterodimeric complex has shown itself amenable to physiologicalregulation by exogenous agents that can readily and effectively alterparacellular permeability in mucosal epithelia. On such agent that hasbeen extensively studied is the bacterial toxin from Vibrio choleraeknown as the “zonula occludens toxin” (ZOT). This toxin mediatesincreased intestinal mucosal permeability and causes disease symptomsincluding diarrhea in infected subjects. Fasano, et al, Proc. Nat. Acad.Sci., U.S.A. 8:5242-5246, 1991; Johnson, et al., J. Clin. Microb.31/3:732-733, 1993; and Karasawa, et al., FEBS Let. 106:143-146, 1993,each incorporated herein by reference. When tested on rabbit ilealmucosa, ZOT increased the intestinal permeability by modulating thestructure of intercellular tight junctions. More recently, it has beenfound that ZOT is capable of reversibly opening tight junctions in theintestinalmucosa. WO 96/37196; U.S. Pat. Nos. 5,945,510; 5,948,629;5,912,323; 5,864,014; 5,827,534; 5,665,389, each incorporated herein byreference. It has also been reported that ZOT is capable of reversiblyopening tight junctions in the nasal mucosa. U.S. Pat. No. 5,908,825,incorporated herein by reference. Thus, ZOT and other agents thatmodulate the ZO1-ZO2 complex will be combinatorially formulated orcoordinately administered with one or more JAM, occludin and claudinpeptides, proteins, analogs and mimetics, and/or other biologicallyactive agents disclosed herein.

Within the methods and compositions of the invention, ZOT, as well asvarious analogs and mimetics of ZOT that function as agonists orantagonists of ZOT activity, are useful for enhancing intranasaldelivery of biologically active agents—by increasing paracellularabsorption into and across the nasal mucosa. In this context, ZOTtypically acts by causing a structural reorganization of tight junctionsmarked by altered localization of the junctional protein ZO1. Withinthese aspects of the invention, ZOT is coordinately administered orcombinatorially formulated with the biologically active agent in aneffective amount to yield significantly enhanced absorption of theactive agent, by reversibly increasing nasal mucosal permeabilitywithout substantial adverse side effects

Suitable methods for determining ZOT biological activity may be selectedfrom a variety of known assays, e.g., involving assaying for a decreaseof tissue or cell culture resistance (Rt) using Ussing chambers (e.g.,as described by Fasano, et al., Proc. Natl. Acad. Sci., USA 8:5242-5246,1991, incorporated herein by reference), assaying for a decrease oftissue resistance (Rt) of intestinal epithelial cell monolayers in Usingchambers; or directly assaying enhancement of absorption of atherapeutic agent across a mucosal surface in vivo.

In addition to ZOT, various other tight junction modulatory agents canbe employed within the methods and compositions of the invention thatmimic the activity of ZOT by reversibly increasing mucosal epithelialparacellular permeability. These include specific binding or blockingagents, such as antibodies, antibody fragments, peptides, peptidemimetics, bacterial toxins and other agents that serve as agonists orantagonists of ZOT activity, or which otherwise alter physiology of theZO1-ZO2 complex (e.g., by blocking dimerization). Naturally, theseadditional regulatory agents include peptide analogs, includingsite-directed mutant variants, of the native ZOT protein, as well astruncated active forms of the protein and peptide mimetics that modelfunctional domains or active sites of the native protein. In addition,these agents include a native mammalian protein “zonulin”, which hasbeen proposed to be an endogenous regulator of tight junctionalphysiology similar in both structural and functional aspects to ZOT(see, e.g., WO 96/37196; WO 00/07609; U.S. Pat. Nos. 5,945,510;5,948,629; 5,912,323; 5,864,014; 5,827,534; 5,665,389, each incorporatedherein by reference), which therefore suggests that ZOT is a convergentevolutionary development of Vibrio cholerae patterned after theendogenous mammalian zonulin regulatory mechanism to facilitate hostentry. Both zonulin and ZOT are proposed to bind a specific membranereceptor, designated “ZOT receptor” (see, e.g., U.S. Pat. Nos.5,864,014; 5,912,323; and 5,948,629, each incorporated herein byreference), which can be used within the invention to screen foradditional agonists and antagonists to ZOT and zonulin activity forregulation of tight junctional physiology. In this context,structure-function analysis of the ZOT receptor, and comparisons betweenZOT and zonulin, will guide production and selection of specific bindingor blocking agents, (e.g., antibodies, antibody fragments, peptides,peptide mimetics, additional bacterial toxins and other agents) to serveas ZOT or zonulin agonists or antagonists, for example with respect toZOT or zonulin binding or activation of the ZOT receptor, to regulatetight junctional physiology within the methods and compositions of theinvention.

Vasodilator Agents and Methods

Yet another class of absorption-promoting agents that shows beneficialutility within the coordinate administration and combinatorialformulation methods and compositions of the invention are vasoactivecompounds, more specifically vasodilators. These compounds functionwithin the invention to modulate the structure and physiology of thesubmucosal vasculature, increasing the transport rate of peptide YYproteins, analogs and mimetics, and other biologically active agentsinto or through the mucosal epithelium and/or to specific target tissuesor compartments (e.g., the systemic circulation or central nervoussystem.).

Vasodilator agents for use within the invention typically causesubmucosal blood vessel relaxation by either a decrease in cytoplasmiccalcium, an increase in nitric oxide (NO) or by inhibiting myosin lightchain kinase. They are generally divided into 9 classes: calciumantagonists, potassium channel openers, ACE inhibitors, angiotensin-IIreceptor antagonists, α-adrenergic and imidazole receptor antagonists,β1-adrenergic agonists, phosphodiesterase inhibitors, eicosanoids and NOdonors.

Despite chemical differences, the pharmacokinetic properties of calciumantagonists are similar. Absorption into the systemic circulation ishigh, and these agents therefore undergo considerable first-passmetabolism by the liver, resulting in individual variation inpharmacokinetics. Except for the newer drugs of the dihydropyridine type(amlodipine, felodipine, isradipine, nilvadipine, nisoldipine andnitrendipine), the half-life of calcium antagonists is short. Therefore,to maintain an effective drug concentration for many of these mayrequire delivery by multiple dosing, or controlled release formulations,as described elsewhere herein. Treatment with the potassium channelopener minoxidil may also be limited in manner and level ofadministration due to potential adverse side effects.

ACE inhibitors prevent conversion of angiotensin-I to angiotensin-II,and are most effective when renin production is increased. Since ACE isidentical to kininase-II, which inactivates the potent endogenousvasodilator bradykinin, ACE inhibition causes a reduction in bradykinindegradation. ACE inhibitors provide the added advantage ofcardioprotective and cardioreparative effects, by preventing andreversing cardiac fibrosis and ventricular hypertrophy in animal models.The predominant elimination pathway of most ACE inhibitors is via renalexcretion. Therefore, renal impairment is associated with reducedelimination and a dosage reduction of 25 to 50% is recommended inpatients with moderate to severe renal impairment.

With regard to NO donors, these compounds are particularly useful withinthe invention for their additional effects on mucosal permeability. Inaddition to the above-noted NO donors, complexes of NO with nucleophilescalled NO/nucleophiles, or NONOates, spontaneously and nonenzymaticallyrelease NO when dissolved in aqueous solution at physiologic pH.Cornfield, et al., J. Lab. Clin. Med. 134:419-425, 1999, incorporatedherein by reference. In contrast, nitro vasodilators such asnitroglycerin require specific enzyme activity for NO release. NONOatesrelease NO with a defined stoichiometry and at predictable rates rangingfrom <3 minutes for diethylamine/NO to approximately 20 hours fordiethylenetriamine/NO (DETANO).

Within certain methods and compositions of the invention, a selectedvasodilator agent is coordinately administered (e.g., systemically orintranasally, simultaneously or in combinatorially effective temporalassociation) or combinatorially formulated with one or more peptide YYproteins, analogs and mimetics, and other biologically active agent(s)in an amount effective to enhance the mucosal absorption of the activeagent(s) to reach a target tissue or compartment in the subject (e.g.,the liver, hepatic portal vein, CNS tissue or fluid, or blood plasma).

Selective Transport-Enhancing Agents and Methods

Within certain aspects of the invention, mucosal delivery ofbiologically active agents is enhanced by methods and agents that targetselective transport mechanisms and promote endo- or transcytocis ofmacromoloecular drugs. In this regard, the compositions and deliverymethods of the invention optionally incorporate a selectivetransport-enhancing agent that facilitates transport of one or morebiologically active agents. These transport-enhancing agents may beemployed in a combinatorial formulation or coordinate administrationprotocol with one or more of the peptide YY proteins, analogs andmimetics disclosed herein, to coordinately enhance delivery of one ormore additional biologically active agent(s) across mucosal transportbarriers, to enhance mucosal delivery of the active agent(s) to reach atarget tissue or compartment in the subject (e.g., the mucosalepithelium, liver, CNS tissue or fluid, or blood plasma). Alternatively,the transport-enhancing agents may be employed in a combinatorialformulation or coordinate administration protocol to directly enhancemucosal delivery of one or more of the peptide YY proteins, analogs andmimetics, with or without enhanced delivery of an additionalbiologically active agent.

Exemplary selective transport-enhancing agents for use within thisaspect of the invention include, but are not limited to, glycosides,sugar-containing molecules, and binding agents such as lectin bindingagents, which are known to interact specifically with epithelialtransport barrier components. Goldstein, et al., Annu. Rev. Cell. Biol.1:1-39, 1985, incorporated herein by reference. For example, specific“bioadhesive” ligands, including various plant and bacterial lectins,which bind to cell surface sugar moieties by receptor-mediatedinteractions can be employed as carriers or conjugated transportmediators for enhancing mucosal, e.g., nasal delivery of biologicallyactive agents within the invention. Certain bioadhesive ligands for usewithin the invention will mediate transmission of biological signals toepithelial target cells that trigger selective uptake of the adhesiveligand by specialized cellular transport processes (endocytosis ortranscytosis). These transport mediators can therefore be employed as a“carrier system” to stimulate or direct selective uptake of one or morepeptide YY proteins, analogs and mimetics, and other biologically activeagent(s) into and/or through mucosal epithelia. These and otherselective transport-enhancing agents significantly enhance mucosaldelivery of macromolecular biopharmaceuticals (particularly peptides,proteins, oligonucleotides and polynucleotide vectors) within theinvention. To utilize these transport-enhancing agents, general carrierformulation and/or conjugation methods as described elsewhere herein areused to coordinately administer a selective transport enhancer (e.g., areceptor-specific ligand) and a biologically active agent to a mucosalsurface, whereby the transport-enhancing agent is effective to triggeror mediate enhanced endo- or transcytosis of the active agent into oracross the mucosal epithelium and/or to additional target cell(s),tissue(s) or compartment(s).

Lectins are plant proteins that bind to specific sugars found on thesurface of glycoproteins and glycolipids of eukaryotic cells.Concentrated solutions of lectins have a ‘mucotractive’ effect, andvarious studies have demonstrated rapid receptor mediated endocytocis(RME) of lectins and lectin conjugates (e.g., concanavalin A conjugatedwith colloidal gold particles) across mucosal surfaces. Additionalstudies have reported that the uptake mechanisms for lectins can beutilized for intestinal drug targeting in vivo. In certain of thesestudies, polystyrene nanoparticles (500 nm) were covalently coupled totomato lectin and reported yielded improved systemic uptake after oraladministration to rats.

In addition to plant lectins, microbial adhesion and invasion factorsprovide a rich source of candidates for use as adhesive/selectivetransport carriers within the mucosal delivery methods and compositionsof the invention. Lehr, Crit. Rev. Therap. Drug Carrier Syst.11:177-218, 1995; Swann, P. A., Pharmaceutical Research 15:826-832,1998, each incorporated herein by reference. Two components arenecessary for bacterial adherence processes, a bacterial ‘adhesin’(adherence or colonization factor) and a receptor on the host cellsurface. Bacteria causing mucosal infections need to penetrate the mucuslayer before attaching themselves to the epithelial surface. Thisattachment is usually mediated by bacterial fimbriae or pilusstructures, although other cell surface components may also take part inthe process. Adherent bacteria colonize mucosal epithelia bymultiplication and initiation of a series of biochemical reactionsinside the target cell through signal transduction mechanisms (with orwithout the help of toxins). Associated with these invasive mechanisms,a wide diversity of bioadhesive proteins (e.g., invasin, internalin)originally produced by various bacteria and viruses are known. Theseallow for extracellular attachment of such microorganisms with animpressive selectivity for host species and even particular targettissues. Signals transmitted by such receptor-ligand interactionstrigger the transport of intact, living microorganisms into, andeventually through, epithelial cells by endo- and transcytoticprocesses. Such naturally occurring phenomena may be harnessed (e.g., bycomplexing biologically active agents such as peptide YY with anadhesin) according to the teachings herein for enhanced delivery ofbiologically active compounds into or across mucosal epithelia and/or toother designated target sites of drug action. One advantage of thisstrategy is that the selective carrier partners thus employed aresubstrate-specific, leaving the natural barrier function of epithelialtissues intact against other solutes. Lehr, Drug Absorption Enhancement,de Boer, Ed., Harwood Academic Publishers, 1994, pp. 325-362,incorporated herein by reference.

Various bacterial and plant toxins that bind epithelial surfaces in aspecific, lectin-like manner are also useful within the methods andcompositions of the invention. For example, diptheria toxin (DT) entershost cells rapidly by RME. Likewise, the B subunit of the E. coli heatlabile toxin binds to the brush border of intestinal epithelial cells ina highly specific, lectin-like manner. Uptake of this toxin andtranscytosis to the basolateral side of the enterocytes has beenreported in vivo and in vitro. Other researches have expressed thetransmembrane domain of diphtheria toxin in E. coli as a maltose-bindingfusion protein and coupled it chemically to high-Mw poly-L-lysine. Theresulting complex was successfully used to mediate internalization of areporter gene in vitro. In addition to these examples, Staphylococcusaureus produces a set of proteins (e.g., staphylococcal enterotoxin A(SEA), SEB, toxic shock syndrome toxin 1 (TSST-1) which act both assuperantigens and toxins. Studies relating to these proteins havereported dose-dependent, facilitated transcytosis of SEB and TSST-1 inCaco-2 cells.

Various plant toxins, mostly ribosome-inactivating proteins (RIPs), havebeen identified that bind to any mammalian cell surface expressinggalactose units and are subsequently internalized by RME. Toxins such asnigrin b, α-sarcin, ricin and saporin, viscumin, and modeccin are highlytoxic upon oral administration (i.e., are rapidly internalized).Therefore, modified, less toxic subunits of these compounds will beuseful within the invention to facilitate the uptake of biologicallyactive agents, including peptide YY proteins, analogs and mimetics.

Viral haemaglutinins comprise another type of transport agent tofacilitate mucosal delivery of biologically active agents within themethods and compositions of the invention. The initial step in manyviral infections is the binding of surface proteins (haemaglutinins) tomucosal cells. These binding proteins have been identified for mostviruses, including rotaviruses, varicella zoster virus, semliki forestvirus, adenoviruses, potato leafroll virus, and reovirus. These andother exemplary viral hemaglutinins can be employed in a combinatorialformulation (e.g., a mixture or conjugate formulation) or coordinateadministration protocol with one or more of the peptide YY proteins,analogs and mimetics disclosed herein, to coordinately enhance mucosaldelivery of one or more additional biologically active agent(s).Alternatively, viral hemaglutinins can be employed in a combinatorialformulation or coordinate administration protocol to directly enhancemucosal delivery of one or more of the peptide YY proteins, analogs andmimetics, with or without enhanced delivery of an additionalbiologically active agent.

A variety of endogenous, selective transport-mediating factors are alsoavailable for use within the invention. Mammalian cells have developedan assortment of mechanisms to facilitate the internalization ofspecific substrates and target these to defined compartments.Collectively, these processes of membrane deformations are termed‘endocytosis’ and comprise phagocytosis, pinocytosis, receptor-mediatedendocytosis (clathrin-mediated RME), and potocytosis(non-clathrin-mediated RME). RME is a highly specific cellular biologicprocess by which, as its name implies, various ligands bind to cellsurface receptors and are subsequently internalized and traffickedwithin the cell. In many cells the process of endocytosis is so activethat the entire membrane surface is internalized and replaced in lessthan a half hour.

RME is initiated when specific ligands bind externally oriented membranereceptors. Binding occurs quickly and is followed by membraneinvagination until an internal vesicle forms within the cell (the earlyendosome, “receptosome,” or CURL (compartment of uncoupling receptor andligand). Localized membrane proteins, lipids and extracellular solutesare also internalized during this process. When the ligand binds to itsspecific receptor, the ligand-receptor complex accumulates in coatedpits. Coated pits are areas of the membrane with high concentration ofendocellular clathrin subunits. The assembly of clathrin molecules onthe coated pit is believed to aid the invagination process. Specializedcoat proteins called adaptins, trap specific membrane receptors thatmove laterally through the membrane in the coated pit area by binding toa signal sequence (Tyr-X-Arg-Phe, where X=any amino acid) at theendocellular carboxy terminus of the receptor. This process ensures thatthe correct receptors are concentrated in the coated pit areas andminimizes the amount of extracellular fluid that is taken up in thecell.

Following the internalization process, the clathrin coat is lost throughthe help of chaperone proteins, and proton pumps lower the endosomal pHto approximately 5.5, which causes dissociation of the receptor-ligandcomplex. CURL serves as a compartment to segregate the recyclingreceptor (e.g., transferrin) from receptor involved in transcytosis(e.g., transcoba-lamin). Endosomes may then move randomly or bysaltatory motion along the microtubules until they reach the trans-Golgireticulum where they are believed to fuse with Golgi components or othermembranous compartments and convert into tubulovesicular complexes andlate endosomes or multivesicular bodies. The fate of the receptor andligand are determined in these sorting vesicles. Some ligands andreceptors are returned to the cell surface where the ligand is releasedinto the extracellular milieu and the receptor is recycled.Alternatively, the ligand is directed to lysosomes for destruction whilethe receptor is recycled to the cell membrane. The endocytotic recyclingpathways of polarized epithelial cells are generally more complex thanin non-polarized cells. In these enterocytes a common recyclingcompartment exists that receives molecules from both apical andbasolateral membranes and is able to correctly return them to theappropriate membrane or membrane recycling compartment.

Current understanding of RME receptor structure and relatedstructure-function relationships has been significantly enhanced by thecloning of mRNA sequences coding for endocytotic receptors. Most RMEreceptors share principal structural features, such as an extracellularligand binding site, a single hydrophobic transmembrane domain (unlessthe receptor is expressed as a dimer), and a cytoplasmic tail encodingendocytosis and other functional signals. Two classes of receptors areproposed based on their orientation in the cell membrane; the aminoterminus of Type I receptors is located on the extracellular side of themembrane, whereas Type II receptors have this same protein tail in theintracellular milieu.

As noted above, potocytosis, or non-clathrin coated endocytosis, takesplace through caveolae, which are uniform omega- or flask-shapedmembrane invaginations 50-80 nm in diameter. This process was firstdescribed as the internalization mechanism of the vitamin folic acid.Morphological studies have implicated caveolae in (i) the transcytosisof macromolecules across endothelial cells; (ii) the uptake of smallmolecules via potocytosis involving GPI-linked receptor molecules and anunknown anion transport protein; (iii) interactions with the actin-basedcytoskeleton; and (iv) the compartmentalization of certain signalingmolecules involved in signal transduction, including G-protein coupledreceptors. Caveolae are characterized by the presence of an integral22-kDa membrane protein termed VIP21-caveolin, which coats thecytoplasmic surface of the membrane. From a drug delivery standpoint,the advantage of potocytosis pathways over clathrin-coated RME pathwayslies in the absence of the pH lowering step, which circumvents theendosomal/lysosomal pathway. This pathway for selectivetransporter-mediated delivery of biologically active agents is thereforeparticularly effective for enhanced delivery of pH-sensitivemacromolecules.

Exemplary among potocytotic transport carriers mechanisms for use withinthe invention is the folate carrier system, which mediates transport ofthe vitamin folic acid (FA) into target cells via specific binding tothe folate receptor (FR). Reddy, et al., Crit. Rev. Ther. Drug Car.Syst. 15:587-627, 1998, incorporated herein by reference. The cellularuptake of free folic acid is mediated by the folate receptor and/or thereduced folate carrier. The folate receptor is aglycosylphosphatidylinositol (GPI)-anchored 38 kDa glycoproteinclustered in caveolae mediating cell transport by potocytosis. While theexpression of the reduced folate carrier is ubiquitously distributed ineukaryotic cells, the folate receptor is principally overexpressed inhuman tumors. Two homologous isoforms (α and β) of the receptor havebeen identified in humans. The α-isoform is found to be frequentlyoverexpressed in epithelial tumors, whereas the β-form is often found innon-epithelial lineage tumors. Consequently, this receptor system hasbeen used in drug-targeting approaches to cancer cells, but also inprotein delivery, gene delivery, and targeting of antisenseoligonucleotides to a variety of cell types.

Folate-drug conjugates are well suited for use within the mucosaldelivery methods of the invention, because they allow penetration oftarget cells exclusively via FR-mediated endocytosis. When FA iscovalently linked, for example, via its γ-carboxyl to a biologicallyactive agent, FR binding affinity (KD˜10⁻¹⁰M) is not significantlycompromised, and endocytosis proceeds relatively unhindered, promotinguptake of the attached active agent by the FR-expressing cell. BecauseFRs are significantly overexpressed on a large fraction of human cancercells (e.g., ovarian, lung, breast, endometrial, renal, colon, andcancers of myeloid hematopoietic cells), this methodology allows forselective delivery of a wide range of therapeutic as well as diagnosticagents to tumors. Folate-mediated tumor targeting has been exploited todate for delivery of the following classes of molecules and molecularcomplexes that find use within the invention: (i) protein toxins, (ii)low-molecular-weight chemotherapeutic agents, (iii) radioimaging agents,(iv) MRI contrast agents, (v) radio-therapeutic agents, (vi) liposomeswith entrapped drugs, (vii) genes, (viii) antisense oligonucleotides,(ix) ribozymes, and (x) immunotherapeutic agents. Swann, P. A.,Pharmaceutical Research 15:826-832, 1998, incorporated herein byreference. In virtually all cases, in vitro studies demonstrate asignificant improvement in potency and/or cancer-cell specificity overthe nontargeted form of the same pharmaceutical agent.

In addition to the folate receptor pathway, a variety of additionalmethods to stimulate transcytosis within the invention are directed tothe transferrin receptor pathway, and the riboflavin receptor pathway.In one aspect, conjugation of a biologically active agent to riboflavincan effectuate RME-mediated uptake. Yet additional embodiments of theinvention utilize vitamin B12 (cobalamin) as a specialized transportprotein (e.g., conjugation partner) to facilitate entry of biologicallyactive agents into target cells. Certain studies suggest that thisparticular system can be employed for the intestinal uptake ofluteinizing hormone releasing factor (LHRH)-analogs, granulocyte colonystimulating factor (G-CSF, 18.8 kDa), erythropoietin (29.5 kDa),α-interferon, and the LHRH-antagonist ANTIDE.

Still other embodiments of the invention utilize transferrin as acarrier or stimulant of RME of mucosally delivered biologically activeagents. Transferrin, an 80 kDa iron-transporting glycoprotein, isefficiently taken up into cells by RME. Transferrin receptors are foundon the surface of most proliferating cells, in elevated numbers onerythroblasts and on many kinds of tumors. According to currentknowledge of intestinal iron absorption, transferrin is excreted intothe intestinal lumen in the form of apotransferrin and is highly stableto attacks from intestinal peptidases. In most cells, diferrictransferrin binds to transferrin receptor (TfR), a dimeric transmembraneglycoprotein of 180 kDa, and the ligand-receptor complex is endocytosedwithin clathrin-coated vesicles. After acidification of these vesicles,iron dissociates from the transferrin/TfR complex and enters thecytoplasm, where it is bound by ferritin (Fn). Recent reports suggestthat insulin covalently coupled to transferrin, is transported acrossCaco-2 cell monolayers by RME. Other studies suggest that oraladministration of this complex to streptozotocin-induced diabetic micesignificantly reduces plasma glucose levels (˜28%), which is furtherpotentiated by BFA pretreatment. The transcytosis of transferrin (Tf)and transferrin conjugates is reportedly enhanced in the presence ofBrefeldin A (BFA), a fungal metabolite. In other studies, BFA treatmenthas been reported to rapidly increase apical endocytosis of both ricinand HRP in MDCK cells. Thus, BFA and other agents that stimulatereceptor-mediated transport can be employed within the methods of theinvention as combinatorially formulated (e.g., conjugated) and/orcoordinately administered agents to enhance receptor-mediated transportof biologically active agents, including peptide YY proteins, analogsand mimetics.

Immunoglobulin transport mechanisms provide yet additional endogenouspathways and reagents for incorporation within the mucosal deliverymethods and compositions of the invention. Receptor-mediatedtranscytosis of immunoglobulin G (IgG) across the neonatal smallintestine serves to convey passive immunity to many newborn mammals. Inrats, IgG in milk selectively binds to neonatal Fc receptors (FcRn)expressed on the surface of the proximal small intestinal enterocytesduring the first three weeks after birth. FcRn binds IgG in apH-dependent manner, with binding occurring at the luminal pH (approx.6-6.5) of the jejunum and release at the pH of plasma (approx. 7.4). TheFc receptor resembles the major histocompatibility complex (MHC) class Iantigens in that it consists of two subunits, a transmembraneglycoprotein (gp50) in association with β₂-microglobulin. In matureabsorptive cells both subunits are colocalized in each of the membranecompartments that mediate transcytosis of IgG. IgG administered in situapparently causes both subunits to concentrate within endocytic pits ofthe apical plasma membrane, suggesting that ligand causes redistributionof receptors at this site. These results support a model for transportin which IgG is transferred across the cell as a complex with bothsubunits.

Methods and compositions are described for the delivery of a therapeuticantigen conjugated to a neonatal Fc receptor (FcR) binding partner tointestinal epithelium, mucosal epithelium or epithelium of the lung. Themethods and compositions are useful as antigen to neonatal FcR bindingpartner conjugates in an oral formulation, aerosol formulation or anasal formulation to deliver drugs and vaccines. U.S. Pat. No.6,485,726, incorporated herein by reference.

Within the methods and compositions of the present invention, IgG andother immune system-related carriers (including polyclonal andmonoclonal antibodies and various fragments thereof) can be coordinateadministered with biologically active agents to provide for targeteddelivery, typically by receptor-mediated transport, of the biologicallyactive agent. For example, the biologically active agent (includingpeptide YY proteins, analogs and mimetics) may be covalently linked tothe IgG or other immunological active agent or, alternatively,formulated in liposomes or other carrier vehicle which is in turnmodified (e.g., coated or covalently linked) to incorporate IgG or otherimmunological transport enhancer. In certain embodiments, polymeric IgAand/or IgM transport agents are employed, which bind to the polymericimmunoglobulin receptors (pIgRs) of target epithelial cells. Withinthese methods, expression of pIgR can be enhanced by cytokines.

Within more detailed aspects of the invention, antibodies and otherimmunological transport agents may be themselves modified for enhancedmucosal delivery, for example, as described in detail elsewhere herein,antibodies may be more effectively administered within the methods andcompositions of the invention by charge modifying techniques. In onesuch aspect, an antibody drug delivery strategy involving antibodycationization is utilized that facilitates both trans-endothelialmigration and target cell endocytosis. Pardridge, et al., JPET286:548-544, 1998, incorporated herein by reference. In one suchstrategy, the pI of the antibody is increased by converting surfacecarboxyl groups of the protein to extended primary amino groups. Thesecationized homologous proteins have no measurable tissue toxicity andhave minimal immunogenicity. In addition, monoclonal antibodies may becationized with retention of affinity for the target protein.

Additional selective transport-enhancing agents for use within theinvention comprise whole bacteria and viruses, including geneticallyengineered bacteria and viruses, as well as components of such bacteriaand viruses. Aside from conventional gene delivery vectors (e.g.,adenovirus), this aspect of the invention includes the use of bacterialghosts and subunit constructs, e.g., as described by Huter, et al.,Journal of Controlled Release 61:51-63, 1999, incorporated herein byreference. Bacterial ghosts are non-denatured bacterial cell envelopes,for example as produced by the controlled expression of theplasmid-encoded lysis gene E of bacteriophage PhiXl74 in gram-negativebacteria. Protein E-specific lysis does not cause any physical orchemical denaturation to bacterial surface structures, and bacterialghosts are therefore useful in development of inactivated whole-cellvaccines. Ghosts produced from Actinobacillus pleuropneumoniae,Pasteurella haemolytica and Salmonella sp. have proved successful invaccination experiments. Recombinant bacterial ghosts can be created bythe expression of foreign genes fused to a membrane-targeting sequence,and thus can carry foreign therapeutic peptides and proteins anchored intheir envelope. The fact that bacterial ghosts preserve a native cellwall, including bioadhesive structures like fimbriae of their livingcounterparts, makes them suitable for the attachment to specific targettissues such as mucosal surfaces. Bacterial ghosts have been shown to bereadily taken up by macrophages, thus adhesion of ghosts to specifictissues can be followed by uptake through phagocytes.

In view of the foregoing, a wide variety of ligands involved inreceptor-mediated transport mechanisms are known in the art and can bevariously employed within the methods and compositions of the invention(e.g., as conjugate partners or coordinately administered mediators) toenhance receptor-mediated transport of biologically active agents,including peptide YY proteins, analogs and mimetics, and otherbiologically active agents disclosed herein. Generally, these ligandsinclude hormones and growth factors, bacterial adhesins and toxins,lectins, metal ions and their carriers, vitamins, immunoglobulins, wholeviruses and bacteria or selected components thereof. Exemplary ligandsamong these classes include, for example, peptide YY, neuropeptide Y,pancreatic peptide, calcitonin, prolactin, epidermal growth factor,glucagon, growth hormone, interferon-β, estrogen, lutenizing hormone,platelet derived growth factor, thyroid stimulating hormone, thyroidhormone, cholera toxin, diptheria toxin, E. coli heat labile toxin,Staphylococcal enterotoxins A and B, ricin, saporin, modeccin, nigrin,sarcin, concanavalin A, transcobalantin, catecholamines, transferrin,folate, riboflavin, vitamin B1, low density lipoprotein, maternal IgO,polymeric IgA, adenovirus, vesicular stomatitis virus, Rous sarcomavirus, V. cholerae, Kiebsiella strains, Serratia strains, parainfluenzavirus, respiratory syncytial virus, Varicella zoster, and Enterobacterstrains. Swann, P. A., Pharmaceutical Research 15:826-832, 1998,incorporated herein by reference.

In certain additional embodiments of the invention, membrane-permeablepeptides (e.g., “arginine rich peptides”) are employed to facilitatedelivery of biologically active agents. While the mechanism of action ofthese peptides remains to be fully elucidated, they provide usefuldelivery enhancing adjuncts for use within the mucosal deliverycompositions and methods herein. In one example, a basic peptide derivedfrom human immunodeficiency virus (HIV)-1 Tat protein (e.g., residues48-60) has been reported to translocate effectively through cellmembranes and accumulate in the nucleus, a characteristic which can beutilized for the delivery of exogenous proteins into cells. The sequenceof Tat (GRKKRRQRRRPPQ) (SEQ ID NO: 789) comprises a highly basic andhydrophilic peptide, which contains 6 arginine and 2 lysine residues inits 13 amino acid residues. Various other arginine-rich peptides havebeen identified which have a translocation activity very similar toTat-(48-60). These include such peptides as the D-amino acid- andarginine-substituted Tat-(48-60), the RNA-binding peptides derived fromvirus proteins, such as HIV-1 Rev, and flock house virus coat proteins,and the DNA binding segments of leucine zipper proteins, such ascancer-related proteins c-Fos and c-Jun, and the yeast transcriptionfactor GCN4. Futaki, et al., Journal Biological Chemistry 276:5836-5840,2000, incorporated herein by reference. These peptides reportedly haveseveral arginine residues marking their only identified commonstructural characteristic, suggesting a common internalization mechanismubiquitous to arginine-rich peptides, which is not explained by typicalendocytosis. Using (Arg)n (n=4-16) peptides (SEQ ID NO: 801), Futaki, etal. teach optimization of arginine residues (n˜8) for efficienttranslocation. Recently, methods have been developed for the delivery ofexogenous proteins into living cells with the help of arginine richmembrane-permeable carrier peptides such as HIV-1 Tat- andAntennapedia-(see, Futaki, et al., supra, and references cited therein,incorporated herein by reference). By genetically or chemicallyhybridizing these carrier peptides with biologically active agents asdescribed herein, additional methods and compositions are thus providedwithin the invention to enhance mucosal delivery.

Polymeric Delivery Vehicles and Methods

Within certain aspects of the invention, peptide YY proteins, analogsand mimetics, other biologically active agents disclosed herein, anddelivery-enhancing agents as described above, are, individually orcombinatorially, incorporated within a mucosally (e.g., nasally)administered formulation that includes a biocompatible polymerfunctioning as a carrier or base. Such polymer carriers includepolymeric powders, matrices or microparticulate delivery vehicles, amongother polymer forms. The polymer can be of plant, animal, or syntheticorigin. Often the polymer is crosslinked. Additionally, in thesedelivery systems the biologically active agent (e.g., a peptide YYprotein, analog or mimetic), can be functionalized in a manner where itcan be covalently bound to the polymer and rendered inseparable from thepolymer by simple washing. In other embodiments, the polymer ischemically modified with an inhibitor of enzymes or other agents whichmay degrade or inactivate the biologically active agent(s) and/ordelivery enhancing agent(s). In certain formulations, the polymer is apartially or completely water insoluble but water swellable polymer,e.g., a hydrogel. Polymers useful in this aspect of the invention aredesirably water interactive and/or hydrophilic in nature to absorbsignificant quantities of water, and they often form hydrogels whenplaced in contact with water or aqueous media for a period of timesufficient to reach equilibrium with water. In more detailedembodiments, the polymer is a hydrogel which, when placed in contactwith excess water, absorbs at least two times its weight of water atequilibrium when exposed to water at room temperature. U.S. Pat. No.6,004,583, incorporated herein by reference.

Drug delivery systems based on biodegradable polymers are preferred inmany biomedical applications because such systems are broken down eitherby hydrolysis or by enzymatic reaction into non-toxic molecules. Therate of degradation is controlled by manipulating the composition of thebiodegradable polymer matrix. These types of systems can therefore beemployed in certain settings for long-term release of biologicallyactive agents. Biodegradable polymers such as poly(glycolic acid) (PGA),poly-(lactic acid) (PLA), and poly(D,L-lactic-co-glycolic acid) (PLGA),have received considerable attention as possible drug delivery carriers,since the degradation products of these polymers have been found to havelow toxicity. During the normal metabolic function of the body thesepolymers degrade into carbon dioxide and water. Mehta, et al, J.Control. Rel. 29:375-384, 1994. These polymers have also exhibitedexcellent biocompatibility.

For prolonging the biological activity of peptide YY proteins, analogsand mimetics, and other biologically active agents disclosed herein, aswell as optional delivery-enhancing agents, these agents may beincorporated into polymeric matrices, e.g., polyorthoesters,polyanhydrides, or polyesters. This yields sustained activity andrelease of the active agent(s), e.g., as determined by the degradationof the polymer matrix. Heller, Formulation and Delivery of Proteins andPeptides, Cleland, et al., Eds., ACS Symposium Series 567, WashingtonD.C., 1994, pp. 292-305; Tabata, et al., Pharm. Res. 10:487-496, 1993;and Cohen, et al., Pharm. Res. 8:713-720, 1991, each incorporated hereinby reference. Although the encapsulation of biotherapeutic moleculesinside synthetic polymers may stabilize them during storage anddelivery, the largest obstacle of polymer-based release technology isthe activity loss of the therapeutic molecules during the formulationprocesses that often involve heat, sonication or organic solvents.Tabata, et al., Pharm. Res. 10:487-496, 1993; and Jones, et al, DrugTargeting and Delivery Series, New Delivery Systems for RecombinantProteins—Practical Issues from Proof of Concept to Clinic 4: 57-67; Lee,et al., Eds., Harwood Academic Publishers, 1995, each incorporatedherein by reference.

Absorption-promoting polymers contemplated for use within the inventionmay include derivatives and chemically or physically modified versionsof the foregoing types of polymers, in addition to other naturallyoccurring or synthetic polymers, gums, resins, and other agents, as wellas blends of these materials with each other or other polymers, so longas the alterations, modifications or blending do not adversely affectthe desired properties, such as water absorption, hydrogel formation,and/or chemical stability for useful application. In more detailedaspects of the invention, polymers such as nylon, acrylan and othernormally hydrophobic synthetic polymers may be sufficiently modified byreaction to become water swellable and/or form stable gels in aqueousmedia.

Suitable polymers for use within the invention should generally bestable alone and in combination with the selected biologically activeagent(s) and additional components of a mucosal formulation, and formstable hydrogels in a range of pH conditions from about pH 1 to pH 10.More typically, they should be stable and form polymers under pHconditions ranging from about 3 to 9, without additional protectivecoatings. However, desired stability properties may be adapted tophysiological parameters characteristic of the targeted site of delivery(e.g., nasal mucosa or secondary site of delivery such as the systemiccirculation). Therefore, in certain formulations higher or lowerstabilities at a particular pH and in a selected chemical or biologicalenvironment will be more desirable.

Absorption-promoting polymers of the invention may include polymers fromthe group of homo- and copolymers based on various combinations of thefollowing vinyl monomers: acrylic and methacrylic acids, acrylamide,methacrylamide, hydroxyethylacrylate or methacrylate, vinylpyrrolidones,as well as polyvinylalcohol and its co- and terpolymers,polyvinylacetate, its co- and terpolymers with the above listed monomersand 2-acrylamido-2-methyl-propanesulfonic acid (AMPS®). Very useful arecopolymers of the above listed monomers with copolymerizable functionalmonomers such as acryl or methacryl amide acrylate or methacrylateesters where the ester groups are derived from straight or branchedchain alkyl, aryl having up to four aromatic rings which may containalkyl substituents of 1 to 6 carbons; steroidal, sulfates, phosphates orcationic monomers such as N,N-dimethylaminoalkyl(meth)acrylamide,dimethylaminoalkyl(meth)acrylate, (meth)acryloxyalkyltrimethylammoniumchloride, (meth)acryloxyalkyldimethylbenzyl ammonium chloride.

Additional absorption-promoting polymers for use within the inventionare those classified as dextrans, dextrins, and from the class ofmaterials classified as natural gums and resins, or from the class ofnatural polymers such as processed collagen, chitin, chitosan, pullalan,zooglan, alginates and modified alginates such as “Kelcoloid” (apolypropylene glycol modified alginate) gellan gums such as “Kelocogel,”Xanathan gums such as “Keltrol,” estastin, alpha hydroxy butyrate andits copolymers, hyaluronic acid and its derivatives, polylactic andglycolic acids.

A very useful class of polymers applicable within the instant inventionare olefinically-unsaturated carboxylic acids containing at least oneactivated carbon-to-carbon olefinic double bond, and at least onecarboxyl group; that is, an acid or functional group readily convertedto an acid containing an olefinic double bond which readily functions inpolymerization because of its presence in the monomer molecule, eitherin the alpha-beta position with respect to a carboxyl group, or as partof a terminal methylene grouping. Olefinically-unsaturated acids of thisclass include such materials as the acrylic acids typified by theacrylic acid itself, alpha-cyano acrylic acid, beta methylacrylic acid(crotonic acid), alpha-phenyl acrylic acid, beta-acryloxy propionicacid, cinnamic acid, p-chloro cinnamic acid, 1-carboxy-4-phenylbutadiene-1,3, itaconic acid, citraconic acid, mesaconic acid,glutaconic acid, aconitic acid, maleic acid, fumaric acid, andtricarboxy ethylene. As used herein, the term “carboxylic acid” includesthe polycarboxylic acids and those acid anhydrides, such as maleicanhydride, wherein the anhydride group is formed by the elimination ofone molecule of water from two carboxyl groups located on the samecarboxylic acid molecule.

Representative acrylates useful as absorption-promoting agents withinthe invention include methyl acrylate, ethyl acrylate, propyl acrylate,isopropyl acrylate, butyl acrylate, isobutyl acrylate, methylmethacrylate, methyl ethacrylate, ethyl methacrylate, octyl acrylate,heptyl acrylate, octyl methacrylate, isopropyl methacrylate,2-ethylhexyl methacrylate, nonyl acrylate, hexyl acrylate, n-hexylmethacrylate, and the like. Higher alkyl acrylic esters are decylacrylate, isodecyl methacrylate, lauryl acrylate, stearyl acrylate,behenyl acrylate and melissyl acrylate and methacrylate versionsthereof. Mixtures of two or three or more long chain acrylic esters maybe successfully polymerized with one of the carboxylic monomers. Othercomonomers include olefins, including alpha olefins, vinyl ethers, vinylesters, and mixtures thereof.

Other vinylidene monomers, including the acrylic nitriles, may also beused as absorption-promoting agents within the methods and compositionsof the invention to enhance delivery and absorption of one or morepeptide YY proteins, analogs and mimetics, and other biologically activeagent(s), including to enhance delivery of the active agent(s) to atarget tissue or compartment in the subject (e.g., the liver, hepaticportal vein, CNS tissue or fluid, or blood plasma). Useful alpha,beta-olefinically unsaturated nitriles are preferably monoolefinicallyunsaturated nitriles having from 3 to 10 carbon atoms such asacrylonitrile, methacrylonitrile, and the like. Most preferred areacrylonitrile and methacrylonitrile. Acrylic amides containing from 3 to35 carbon atoms including monoolefinically unsaturated amides also maybe used. Representative amides include acrylamide, methacrylamide,N-t-butyl acrylamide, N-cyclohexyl acrylamide, higher alkyl amides,where the alkyl group on the nitrogen contains from 8 to 32 carbonatoms, acrylic amides including N-alkylol amides of alpha,beta-olefinically unsaturated carboxylic acids including those havingfrom 4 to 10 carbon atoms such as N-methylol acrylamide, N-propanolacrylamide, N-methylol methacrylamide, N-methylol maleimide, N-methylolmaleamic acid esters, N-methylol-p-vinyl benzamide, and the like.

Yet additional useful absorption promoting materials are alpha-olefinscontaining from 2 to 18 carbon atoms, more preferably from 2 to 8 carbonatoms; dienes containing from 4 to 10 carbon atoms; vinyl esters andallyl esters such as vinyl acetate; vinyl aromatics such as styrene,methyl styrene and chloro-styrene; vinyl and allyl ethers and ketonessuch as vinyl methyl ether and methyl vinyl ketone; chloroacrylates;cyanoalkyl acrylates such as alpha-cyanomethyl acrylate, and the alpha-,beta-, and gamma-cyanopropyl acrylates; alkoxyacrylates such as methoxyethyl acrylate; haloacrylates as chloroethyl acrylate; vinyl halides andvinyl chloride, vinylidene chloride and the like; divinyls, diacrylatesand other polyfunctional monomers such as divinyl ether, diethyleneglycol diacrylate, ethylene glycol dimethacrylate,methylene-bis-acrylamide, allylpentaerythritol, and the like; andbis(beta-haloalkyl) alkenyl phosphonates such as bis(beta-chloroethyl)vinyl phosphonate and the like as are known to those skilled in the art.Copolymers wherein the carboxy containing monomer is a minorconstituent, and the other vinylidene monomers present as majorcomponents are readily prepared in accordance with the methods disclosedherein.

When hydrogels are employed as absorption promoting agents within theinvention, these may be composed of synthetic copolymers from the groupof acrylic and methacrylic acids, acrylamide, methacrylamide,hydroxyethylacrylate (HEA) or methacrylate (HEMA), and vinylpyrrolidoneswhich are water interactive and swellable. Specific illustrativeexamples of useful polymers, especially for the delivery of peptides orproteins, are the following types of polymers: (meth)acrylamide and 0.1to 99 wt. % (meth)acrylic acid; (meth)acrylamides and 0.1-75 wt %(meth)acryloxyethyl trimethylammonium chloride; (meth)acrylamide and0.1-75 wt % (meth)acrylamide; acrylic acid and 0.1-75 wt %alkyl(meth)acrylates; (meth)acrylamide and 0.1-75 wt % AMPS® (trademarkof Lubrizol Corp.); (meth)acrylamide and 0 to 30 wt %alkyl(meth)acrylamides and 0.1-75 wt % AMPS®; (meth)acrylamide and0.1-99 wt. % HEMA; (metb)acrylamide and 0.1 to 75 wt % HEMA and 0.1 to99% (meth)acrylic acid; (meth)acrylic acid and 0.1-99 wt % HEMA; 50 mole% vinyl ether and 50 mole % maleic anhydride; (meth)acrylamide and 0.1to 75 wt % (meth)acryloxyalky dimethyl benzylammonium chloride;(meth)acrylamide and 0.1 to 99 wt % vinyl pyrrolidone; (meth)acrylamideand 50 wt % vinyl pyrrolidone and 0.1-99.9 wt % (meth)acrylic acid;(meth)acrylic acid and 0.1 to 75 wt % AMPS® and 0.1-75 wt %alkyl(meth)acrylamide. In the above examples, alkyl means C₁ to C₃₀,preferably C₁ to C₂₂, linear and branched and C₄ to C₁₆ cyclic; where(meth) is used, it means that the monomers with and without the methylgroup are included. Other very useful hydrogel polymers are swellable,but insoluble versions of poly(vinyl pyrrolidone) starch, carboxymethylcellulose and polyvinyl alcohol.

Additional polymeric hydrogel materials useful within the inventioninclude (poly) hydroxyalkyl (meth)acrylate: anionic and cationichydrogels: poly(electrolyte) complexes; poly(vinyl alcohols) having alow acetate residual: a swellable mixture of crosslinked agar andcrosslinked carboxymethyl cellulose: a swellable composition comprisingmethyl cellulose mixed with a sparingly crosslinked agar; a waterswellable copolymer produced by a dispersion of finely divided copolymerof maleic anhydride with styrene, ethylene, propylene, or isobutylene; awater swellable polymer of N-vinyl lactams; swellable sodium salts ofcarboxymethyl cellulose; and the like.

Other gelable, fluid imbibing and retaining polymers useful for formingthe hydrophilic hydrogel for mucosal delivery of biologically activeagents within the invention include pectin; polysaccharides such asagar, acacia, karaya, tragacenth, algins and guar and their crosslinkedversions; acrylic acid polymers, copolymers and salt derivatives,polyacrylamides; water swellable indene maleic anhydride polymers;starch graft copolymers; acrylate type polymers and copolymers withwater absorbability of about 2 to 400 times its original weight;diesters of polyglucan; a mixture of crosslinked poly(vinyl alcohol) andpoly(N-vinyl-2-pyrrolidone); polyoxybutylene-polyethylene blockcopolymer gels; carob gum; polyester gels; poly urea gels; polyethergels; polyamide gels; polyimide gels; polypeptide gels; polyamino acidgels; poly cellulosic gels; crosslinked indene-maleic anhydride acrylatepolymers; and polysaccharides.

Synthetic hydrogel polymers for use within the invention may be made byan infinite combination of several monomers in several ratios. Thehydrogel can be crosslinked and generally possesses the ability toimbibe and absorb fluid and swell or expand to an enlarged equilibriumstate. The hydrogel typically swells or expands upon delivery to thenasal mucosal surface, absorbing about 2-5, 5-10, 10-50, up to 50-100 ormore times fold its weight of water. The optimum degree of swellabilityfor a given hydrogel will be determined for different biologicallyactive agents depending upon such factors as molecular weight, size,solubility and diffusion characteristics of the active agent carried byor entrapped or encapsulated within the polymer, and the specificspacing and cooperative chain motion associated with each individualpolymer.

Hydrophilic polymers useful within the invention are water insoluble butwater swellable. Such water swollen polymers as typically referred to ashydrogels or gels. Such gels may be conveniently produced from watersoluble polymer by the process of crosslinking the polymers by asuitable crosslinking agent. However, stable hydrogels may also beformed from specific polymers under defined conditions of pH,temperature and/or ionic concentration, according to know methods in theart. Typically the polymers are cross-linked, that is, cross-linked tothe extent that the polymers possess good hydrophilic properties, haveimproved physical integrity (as compared to non cross-linked polymers ofthe same or similar type) and exhibit improved ability to retain withinthe gel network both the biologically active agent of interest andadditional compounds for coadministration therewith such as a cytokineor enzyme inhibitor, while retaining the ability to release the activeagent(s) at the appropriate location and time.

Generally hydrogel polymers for use within the invention are crosslinkedwith a difunctional cross-linking in the amount of from 0.01 to 25weight percent, based on the weight of the monomers forming thecopolymer, and more preferably from 0.1 to 20 weight percent and moreoften from 0.1 to 15 weight percent of the crosslinking agent. Anotheruseful amount of a crosslinking agent is 0.1 to 10 weight percent. Tri,tetra or higher multifunctional crosslinking agents may also beemployed. When such reagents are utilized, lower amounts may be requiredto attain equivalent crosslinking density, i.e., the degree ofcrosslinking, or network properties that are sufficient to containeffectively the biologically active agent(s).

The crosslinks can be covalent, ionic or hydrogen bonds with the polymerpossessing the ability to swell in the presence of water containingfluids. Such crosslinkers and crosslinking reactions are known to thoseskilled in the art and in many cases are dependent upon the polymersystem. Thus a crosslinked network may be formed by free radicalcopolymerization of unsaturated monomers. Polymeric hydrogels may alsobe formed by crosslinking preformed polymers by reacting functionalgroups found on the polymers such as alcohols, acids, amines with suchgroups as glyoxal, formaldehyde or glutaraldehyde, bis anhydrides andthe like.

The polymers also may be cross-linked with any polyene, e.g., decadieneor trivinyl cyclohexane; acrylamides, such asN,N-methylene-bis(acrylamide); polyfunctional acrylates, such astrimethylol propane triacrylate; or polyfunctional vinylidene monomercontaining at least 2 terminal CH.sub.2<groups, including, for example,divinyl benzene, divinyl naphthalene, allyl acrylates and the like. Incertain embodiments, cross-linking monomers for use in preparing thecopolymers are polyalkenyl polyethers having more than one alkenyl ethergrouping per molecule, which may optionally possess alkenyl groups inwhich an olefinic double bond is present attached to a terminalmethylene grouping (e.g., made by the etherification of a polyhydricalcohol containing at least 2 carbon atoms and at least 2 hydroxylgroups). Compounds of this class may be produced by reacting an alkenylhalide, such as allyl chloride or allyl bromide, with a stronglyalkaline aqueous solution of one or more polyhydric alcohols. Theproduct may be a complex mixture of polyethers with varying numbers ofether groups. Efficiency of the polyether cross-linking agent increaseswith the number of potentially polymerizable groups on the molecule.Typically, polyethers containing an average of two or more alkenyl ethergroupings per molecule are used. Other cross-linking monomers includefor example, diallyl esters, dimethallyl ethers, allyl or methallylacrylates and acrylamides, tetravinyl silane, polyalkenyl methanes,diacrylates, and dimethacrylates, divinyl compounds such as divinylbenzene, polyallyl phosphate, diallyloxy compounds and phosphite estersand the like. Typical agents are allyl pentaerythritol, allyl sucrose,trimethylolpropane triacrylate, 1,6-hexanediol diacrylate,trimethylolpropane diallyl ether, pentaerythritol triacrylate,tetramethylene dimethacrylate, ethylene diacrylate, ethylenedimethacrylate, triethylene glycol dimethacrylate, and the like. Allylpentaerythritol, trimethylolpropane diallylether and allyl sucroseprovide suitable polymers. When the cross-linking agent is present, thepolymeric mixtures usually contain between about 0.01 to 20 weightpercent, e.g., 1%, 5%, or 10% or more by weight of cross-linking monomerbased on the total of carboxylic acid monomer, plus other monomers.

In more detailed aspects of the invention, mucosal delivery of peptideYY proteins, analogs and mimetics, and other biologically active agentsdisclosed herein, is enhanced by retaining the active agent(s) in aslow-release or enzymatically or physiologically protective carrier orvehicle, for example a hydrogel that shields the active agent from theaction of the degradative enzymes. In certain embodiments, the activeagent is bound by chemical means to the carrier or vehicle, to which mayalso be admixed or bound additional agents such as enzyme inhibitors,cytokines, etc. The active agent may alternately be immobilized throughsufficient physical entrapment within the carrier or vehicle, e.g., apolymer matrix.

Polymers such as hydrogels useful within the invention may incorporatefunctional linked agents such as glycosides chemically incorporated intothe polymer for enhancing intranasal bioavailability of active agentsformulated therewith. Examples of such glycosides are glucosides,fructosides, galactosides, arabinosides, mannosides and their alkylsubstituted derivatives and natural glycosides such as arbutin,phlorizin, amygdalin, digitonin, saponin, and indican. There are severalways in which a typical glycoside may be bound to a polymer. Forexample, the hydrogen of the hydroxyl groups of a glycoside or othersimilar carbohydrate may be replaced by the alkyl group from a hydrogelpolymer to form an ether. Also, the hydroxyl groups of the glycosidesmay be reacted to esterify the carboxyl groups of a polymeric hydrogelto form polymeric esters in situ. Another approach is to employcondensation of acetobromoglucose with cholest-5-en-3beta-ol on acopolymer of maleic acid. N-substituted polyacrylamides can besynthesized by the reaction of activated polymers withomega-aminoalkylglycosides: (1) (carbohydrate-spacer)(n)-polyacrylamide,‘pseudopolysaccharides’; (2) (carbohydratespacer)(n)-phosphatidylethanolamine(m)-polyacrylamide, neoglycolipids,derivatives of phosphatidylethanolamine; (3)(carbohydrate-spacer)(n)-biotin(m)-polyacrylamide. These biotinylatedderivatives may attach to lectins on the mucosal surface to facilitateabsorption of the biologically active agent(s), e.g., apolymer-encapsulated peptide YY.

Within more detailed aspects of the invention, one or more peptide YYproteins, analogs and mimetics, and/or other biologically active agents,disclosed herein, optionally including secondary active agents such asprotease inhibitor(s), cytokine(s), additional modulator(s) ofintercellular junctional physiology, etc., are modified and bound to apolymeric carrier or matrix. For example, this may be accomplished bychemically binding a peptide or protein active agent and other optionalagent(s) within a crosslinked polymer network. It is also possible tochemically modify the polymer separately with an interactive agent suchas a glycosidal containing molecule. In certain aspects, thebiologically active agent(s), and optional secondary active agent(s),may be functionalized, i.e., wherein an appropriate reactive group isidentified or is chemically added to the active agent(s). Most often anethylenic polymerizable group is added, and the functionalized activeagent is then copolymerized with monomers and a crosslinking agent usinga standard polymerization method such as solution polymerization(usually in water), emulsion, suspension or dispersion polymerization.Often, the functionalizing agent is provided with a high enoughconcentration of functional or polymerizable groups to insure thatseveral sites on the active agent(s) are functionalized. For example, ina polypeptide comprising 16 amine sites, it is generally desired tofunctionalize at least 2, 4, 5, 7, and up to 8 or more of the sites.

After functionalization, the functionalized active agent(s) is/are mixedwith monomers and a crosslinking agent that comprise the reagents fromwhich the polymer of interest is formed. Polymerization is then inducedin this medium to create a polymer containing the bound active agent(s).The polymer is then washed with water or other appropriate solvents andotherwise purified to remove trace unreacted impurities and, ifnecessary, ground or broken up by physical means such as by stirring,forcing it through a mesh, ultrasonication or other suitable means to adesired particle size. The solvent, usually water, is then removed insuch a manner as to not denature or otherwise degrade the activeagent(s). One desired method is lyophilization (freeze drying) but othermethods are available and may be used (e.g., vacuum drying, air drying,spray drying, etc.).

To introduce polymerizable groups in peptides, proteins and other activeagents within the invention, it is possible to react available amino,hydroxyl, thiol and other reactive groups with electrophiles containingunsaturated groups. For example, unsaturated monomers containingN-hydroxy succinimidyl groups, active carbonates such as p-nitrophenylcarbonate, trichlorophenyl carbonates, tresylate, oxycarbonylimidazoles,epoxide, isocyanates and aldehyde, and unsaturated carboxymethyl azidesand unsaturated orthopyridyl-disulfide belong to this category ofreagents. Illustrative examples of unsaturated reagents are allylglycidyl ether, allyl chloride, allylbromide, allyl iodide, acryloylchloride, allyl isocyanate, allylsulfonyl chloride, maleic anhydride,copolymers of maleic anhydride and allyl ether, and the like.

All of the lysine active derivatives, except aldehyde, can generallyreact with other amino acids such as imidazole groups of histidine andhydroxyl groups of tyrosine and the thiol groups of cystine if the localenvironment enhances nucleophilicity of these groups. Aldehydecontaining functionalizing reagents are specific to lysine. These typesof reactions with available groups from lysines, cysteines, tyrosinehave been extensively documented in the literature and are known tothose skilled in the art.

In the case of biologically active agents that contain amine groups, itis convenient to react such groups with an acyloyl chloride, such asacryloyl chloride, and introduce the polymerizable acrylic group ontothe reacted agent. Then during preparation of the polymer, such asduring the crosslinking of the copolymer of acrylamide and acrylic acid,the functionalized active agent, through the acrylic groups, is attachedto the polymer and becomes bound thereto.

In additional aspects of the invention, biologically active agents,including peptides, proteins, nucleosides, and other molecules which arebioactive in vivo, are conjugation-stabilized by covalently bonding oneor more active agent(s) to a polymer incorporating as an integral partthereof both a hydrophilic moiety, e.g., a linear polyalkylene glycol, alipophilic moiety (see, e.g., U.S. Pat. No. 5,681,811, incorporatedherein by reference). In one aspect, a biologically active agent iscovalently coupled with a polymer comprising (i) a linear polyalkyleneglycol moiety, and (ii) a lipophilic moiety, wherein the active agent,linear polyalkylene glycol moiety, and the lipophilic moiety areconformationally arranged in relation to one another such that theactive therapeutic agent has an enhanced in vivo resistance to enzymaticdegradation (i.e., relative to its stability under similar conditions inan unconjugated form devoid of the polymer coupled thereto). In anotheraspect, the conjugation-stabilized formulation has a three-dimensionalconformation comprising the biologically active agent covalently coupledwith a polysorbate complex comprising (i) a linear polyalkylene glycolmoiety, and (ii) a lipophilic moiety, wherein the active agent, thelinear polyalkylene glycol moiety and the lipophilic moiety areconformationally arranged in relation to one another such that (a) thelipophilic moiety is exteriorly available in the three-dimensionalconformation, and (b) the active agent in the composition has anenhanced in vivo resistance to enzymatic degradation.

In a further related aspect, a multiligand conjugated complex isprovided which comprises a biologically active agent covalently coupledwith a triglyceride backbone moiety through a polyalkylene glycol spacergroup bonded at a carbon atom of the triglyceride backbone moiety, andat least one fatty acid moiety covalently attached either directly to acarbon atom of the triglyceride backbone moiety or covalently joinedthrough a polyalkylene glycol spacer moiety (see, e.g., U.S. Pat. No.5,681,811, incorporated herein by reference). In such a multiligandconjugated therapeutic agent complex, the alpha′ and beta carbon atomsof the triglyceride bioactive moiety may have fatty acid moietiesattached by covalently bonding either directly thereto, or indirectlycovalently bonded thereto through polyalkylene glycol spacer moieties.Alternatively, a fatty acid moiety may be covalently attached eitherdirectly or through a polyalkylene glycol spacer moiety to the alpha andalpha′ carbons of the triglyceride backbone moiety, with the bioactivetherapeutic agent being covalently coupled with the gamma-carbon of thetriglyceride backbone moiety, either being directly covalently bondedthereto or indirectly bonded thereto through a polyalkylene spacermoiety. It will be recognized that a wide variety of structural,compositional, and conformational forms are possible for the multiligandconjugated therapeutic agent complex comprising the triglyceridebackbone moiety, within the scope of the invention. It is further notedthat in such a multiligand conjugated therapeutic agent complex, thebiologically active agent(s) may advantageously be covalently coupledwith the triglyceride modified backbone moiety through alkyl spacergroups, or alternatively other acceptable spacer groups, within thescope of the invention. As used in such context, acceptability of thespacer group refers to steric, compositional, and end use applicationspecific acceptability characteristics.

In yet additional aspects of the invention, a conjugation-stabilizedcomplex is provided which comprises a polysorbate complex comprising apolysorbate moiety including a triglyceride backbone having covalentlycoupled to alpha, alpha′ and beta carbon atoms thereof functionalizinggroups including (i) a fatty acid group; and (ii) a polyethylene glycolgroup having a biologically active agent or moiety covalently bondedthereto, e.g., bonded to an appropriate functionality of thepolyethylene glycol group. U.S. Pat. No. 5,681,811, incorporated hereinby reference. Such covalent bonding may be either direct, e.g., to ahydroxy terminal functionality of the polyethylene glycol group, oralternatively, the covalent bonding may be indirect, e.g., by reactivelycapping the hydroxy terminus of the polyethylene glycol group with aterminal carboxy functionality spacer group, so that the resultingcapped polyethylene glycol group has a terminal carboxy functionality towhich the biologically active agent or moiety may be covalently bonded.

In yet additional aspects of the invention, a stable, aqueously soluble,conjugation-stabilized complex is provided which comprises one or morepeptide YY proteins, analogs and mimetics, and/or other biologicallyactive agent(s)+disclosed herein covalently coupled to a physiologicallycompatible polyethylene glycol (PEG) modified glycolipid moiety. In suchcomplex, the biologically active agent(s) may be covalently coupled tothe physiologically compatible PEG modified glycolipid moiety by alabile covalent bond at a free amino acid group of the active agent,wherein the labile covalent bond is scissionable in vivo by biochemicalhydrolysis and/or proteolysis. The physiologically compatible PEGmodified glycolipid moiety may advantageously comprise a polysorbatepolymer, e.g., a polysorbate polymer comprising fatty acid ester groupsselected from the group consisting of monopalmitate, dipalmitate,monolaurate, dilaurate, trilaurate, monoleate, dioleate, trioleate,monostearate, distearate, and tristearate. In such complex, thephysiologically compatible PEG modified glycolipid moiety may suitablycomprise a polymer selected from the group consisting of polyethyleneglycol ethers of fatty acids, and polyethylene glycol esters of fattyacids, wherein the fatty acids for example comprise a fatty acidselected from the group consisting of lauric, palmitic, oleic, andstearic acids.

Storage of Material

In certain aspects of the invention, the combinatorial formulationsand/or coordinate administration methods herein incorporate an effectiveamount of peptides and proteins which may adhere to charged glassthereby reducing the effective concentration in the container. Silanizedcontainers, for example, silanized glass containers, are used to storethe finished product to reduce adsorption of the polypeptide or proteinto a glass container.

In yet additional aspects of the invention, a kit for treatment of amammalian subject comprises a stable pharmaceutical composition of oneor more peptide YY compound(s) formulated for mucosal delivery to themammalian subject wherein the composition is effective to alleviate oneor more symptom(s) of obesity, cancer, or malnutrition or wastingrelated to cancer in said subject without unacceptable adverse sideeffects. The kit further comprises a pharmaceutical reagent vial tocontain the one or more peptide YY compounds. The pharmaceutical reagentvial is composed of pharmaceutical grade polymer, glass or othersuitable material. The pharmaceutical reagent vial is, for example, asilanized glass vial. The kit further comprises an aperture for deliveryof the composition to a nasal mucosal surface of the subject. Thedelivery aperture is composed of a pharmaceutical grade polymer, glassor other suitable material. The delivery aperture is, for example, asilanized glass.

A silanization technique combines a special cleaning technique for thesurfaces to be silanized with a silanization process at low pressure.The silane is in the gas phase and at an enhanced temperature of thesurfaces to be silanized. The method provides reproducible surfaces withstable, homogeneous and functional silane layers having characteristicsof a monolayer. The silanized surfaces prevent binding to the glass ofpolypeptides or mucosal delivery enhancing agents of the presentinvention. See WO 83/02669 A1, incorporated herein by reference.

The procedure is useful to prepare silanized pharmaceutical reagentvials to hold peptide YY compositions of the present invention. Glasstrays are cleaned by rinsing with double distilled water (ddH₂O) beforeusing. The silane tray is then be rinsed with 95% EtOH, and the acetonetray is rinsed with acetone. Pharmaceutical reagent vials are sonicatedin acetone for 10 minutes. After the acetone sonication, reagent vialsare washed in ddH₂O tray at least twice. Reagent vials are sonicated in0.1M NaOH for 10 minutes. While the reagent vials are sonicating inNaOH, the silane solution is made under a hood. (Silane solution: 800 mLof 95% ethanol; 96 L of glacial acetic acid; 25 mL ofglycidoxypropyltrimethoxy silane). After the NaOH sonication, reagentvials are washed in ddH₂O tray at least twice. The reagent vials aresonicated in silane solution for 3 to 5 minutes. The reagent vials arewashed in 100% EtOH tray. The reagent vials are dried with prepurifiedN₂ gas and stored in a 100° C. oven for at least 2 hours before using.

Bioadhesive Delivery Vehicles and Methods

In certain aspects of the invention, the combinatorial formulationsand/or coordinate administration methods herein incorporate an effectiveamount of a nontoxic bioadhesive as an adjunct compound or carrier toenhance mucosal delivery of one or more biologically active agent(s).Bioadhesive agents in this context exhibit general or specific adhesionto one or more components or surfaces of the targeted mucosa. Thebioadhesive maintains a desired concentration gradient of thebiologically active agent into or across the mucosa to ensurepenetration of even large molecules (e.g., peptides and proteins) intoor through the mucosal epithelium. Typically, employment of abioadhesive within the methods and compositions of the invention yieldsa two- to five-fold, often a five- to ten-fold increase in permeabilityfor peptides and proteins into or through the mucosal epithelium. Thisenhancement of epithelial permeation often permits effectivetransmucosal delivery of large macromolecules, for example to the basalportion of the nasal epithelium or into the adjacent extracellularcompartments or a blood plasma or CNS tissue or fluid.

This enhanced delivery provides for greatly improved effectiveness ofdelivery of bioactive peptides, proteins and other macromoleculartherapeutic species. These results will depend in part on thehydrophilicity of the compound, whereby greater penetration will beachieved with hydrophilic species compared to water insoluble compounds.In addition to these effects, employment of bioadhesives to enhance drugpersistence at the mucosal surface can elicit a reservoir mechanism forprotracted drug delivery, whereby compounds not only penetrate acrossthe mucosal tissue but also back-diffuse toward the mucosal surface oncethe material at the surface is depleted.

A variety of suitable bioadhesives are disclosed in the art for oraladministration, U.S. Pat. Nos. 3,972,995; 4,259,314; 4,680,323;4,740,365; 4,573,996; 4,292,299; 4,715,369; 4,876,092; 4,855,142;4,250,163; 4,226,848; 4,948,580; U.S. Pat. Reissue No. 33,093; andRobinson, Proc. Intern. Symp. Control. Rel. Bioact. Mater. 18:75, 1991,each incorporated herein by reference, which find use within the novelmethods and compositions of the invention. The potential of variousbioadhesive polymers as a mucosal, e.g., nasal, delivery platform withinthe methods and compositions of the invention can be readily assessed bydetermining their ability to retain and release a specific biologicallyactive agent, e.g., a peptide YY, as well as by their capacity tointeract with the mucosal surfaces following incorporation of the activeagent therein. In addition, well known methods will be applied todetermine the biocompatibility of selected polymers with the tissue atthe site of mucosal administration. One aspect of polymerbiocompatibility is the potential effect for the polymer to induce acytokine response. In certain circumstances, implanted polymers havebeen shown to induce the release of inflammatory cytokines from adheringcells, such as monocytes and macrophages. Similar potential adversereactions of mucosal epithelial cells in contact with candidatebioadhesive polymers will be determined using routine in vitro and invivo assays. Since epithelial cells have the ability to secrete a numberof cytokines, the induction of cytokine responses in epithelial cellswill often provide an adequate measure of biocompatibility of a selectedpolymer delivery platform.

When the target mucosa is covered by mucus (i.e., in the absence ofmucolytic or mucus-clearing treatment), it can serve as a connectinglink to the underlying mucosal epithelium. Therefore, the term“bioadhesive” as used herein also covers mucoadhesive compounds usefulfor enhancing mucosal delivery of biologically active agents within theinvention. However, adhesive contact to mucosal tissue mediated throughadhesion to a mucus gel layer may be limited by incomplete or transientattachment between the mucus layer and the underlying tissue,particularly at nasal surfaces where rapid mucus clearance occurs. Inthis regard, mucin glycoproteins are continuously secreted and,immediately after their release from cells or glands, form aviscoelastic gel. The luminal surface of the adherent gel layer,however, is continuously eroded by mechanical, enzymatic and/or ciliaryaction. Where such activities are more prominent, or where longeradhesion times are desired, the coordinate administration methods andcombinatorial formulation methods of the invention may furtherincorporate mucolytic and/or ciliostatic methods or agents as disclosedherein above.

Bioadhesive and other delivery enhancing agents within the methods andcompositions of the invention can improve the effectiveness of atreatment by helping maintain the drug concentration between effectiveand toxic levels, by inhibiting dilution of the drug away from thedelivery point, and improving targeting and localization of the drug. Inthis context, bioadhesion increases the intimacy and duration of contactbetween a drug-containing polymer and the mucosal surface. The combinedeffects of this enhanced, direct drug absorption, and the decrease inexcretion rate that results from reduced diffusion and improvedlocalization, significantly enhances bioavailability of the drug andallows for a smaller dosage and less frequent administration.

Typically, mucoadhesive polymers for use within the invention arenatural or synthetic macromolecules which adhere to wet mucosal tissuesurfaces by complex, but non-specific, mechanisms. In addition to thesemucoadhesive polymers, the invention also provides methods andcompositions incorporating bioadhesives that adhere directly to a cellsurface, rather than to mucus, by means of specific, includingreceptor-mediated, interactions. One example of bioadhesives thatfunction in this specific manner is the group of compounds known aslectins. These are glycoproteins with an ability to specificallyrecognize and bind to sugar molecules, e.g. glycoproteins orglycolipids, which form part of intranasal epithelial cell membranes andcan be considered as “lectin receptors.”

In various embodiments, the coordinate administration methods of theinvention optionally incorporate bioadhesive materials that yieldprolonged residence time at the mucosal surface. Alternatively, thebioadhesive material may otherwise facilitate mucosal absorption of thebiologically active agent, e.g., by facilitating localization of theactive agent to a selected target site of activity (e.g., the liver, CNStissue or fluid, or blood plasma). In additional aspects, adjunctdelivery or combinatorial formulation of bioadhesive agents within themethods and compositions of the invention intensify contact of thebiologically active agent with the target mucosa, including byincreasing epithelial permeability, (e.g., to effectively increase thedrug concentration gradient). In further alternate embodiments,bioadhesives and other polymers disclosed herein serve to inhibitproteolytic or other enzymes that might degrade the biologically activeagent. For a review of different approaches to bioadhesion that areuseful within the coordinate administration, multi-processing and/orcombinatorial formulation methods and compositions of the invention.Lehr, C. M., Eur J. Drug Metab. Pharmacokinetics, 21:139-148, 1996,incorporated herein by reference.

In certain aspects of the invention, bioadhesive materials for enhancingintranasal delivery of biologically active agents comprise a matrix of ahydrophilic, e.g., water soluble or swellable, polymer or a mixture ofpolymers that can adhere to a wet mucous surface. These adhesives may beformulated as ointments, hydrogels (see above) thin films, and otherapplication forms. Often, these adhesives have the biologically activeagent mixed therewith to effectuate slow release or local delivery ofthe active agent. Some are formulated with additional ingredients tofacilitate penetration of the active agent through the nasal mucosa,e.g., into the circulatory system of the individual.

Various polymers, both natural and synthetic ones, show significantbinding to mucus and/or mucosal epithelial surfaces under physiologicalconditions. The strength of this interaction can readily be measured bymechanical peel or shear tests. A variety of suitable test methods andinstruments to serve such purposes are known in the art. Gu, et al.,Crit. Rev. Ther. Drug Carrier Syst. 5:21-67, 1988; Duchene, et al., DrugDev. Ind. Pharm. 14:283-318, 1988, incorporated herein by reference.When applied to a humid mucosal surface, many dry materials willspontaneously adhere, at least slightly. After such an initial contact,some hydrophilic materials start to attract water by adsorption,swelling or capillary forces, and if this water is absorbed from theunderlying substrate or from the polymer-tissue interface, the adhesionmay be sufficient to achieve the goal of enhancing mucosal absorption ofbiologically active agents. Al-Dujaili, et al., Int. J. Pharm. 34:75-79,1986; Marvola, et al., J. Pharm. Sci. 72:1034-1036, 1983; Marvola, etal., J. Pharm. Sci. 71:975-977, 1982; and Swisher, et al., Int. J.Pharm. 22:219, 1984; Chen, et al., Adhesion in Biological Systems, 172,Manly, Ed., Academic Press, London, 1970, each incorporated herein byreference. Such ‘adhesion by hydration’ can be quite strong, butformulations adapted to employ this mechanism must account for swellingwhich continues as the dosage transforms into a hydrated mucilage. Thisis projected for many hydrocolloids useful within the invention,especially some cellulose-derivatives, which are generally non-adhesivewhen applied in pre-hydrated state. Nevertheless, bioadhesive drugdelivery systems for mucosal administration are effective within theinvention when such materials are applied in the form of a dry polymericpowder, microsphere, or film-type delivery form.

Other polymers adhere to mucosal surfaces not only when applied in dry,but also in fully hydrated state, and in the presence of excess amountsof water. The selection of a mucoadhesive thus requires dueconsideration of the conditions, physiological as well asphysico-chemical, under which the contact to the tissue will be formedand maintained. In particular, the amount of water or humidity usuallypresent at the intended site of adhesion, and the prevailing pH, areknown to largely affect the mucoadhesive binding strength of differentpolymers.

Several polymeric bioadhesive drug delivery systems have been fabricatedand studied in the past 20 years, not always with success. A variety ofsuch carriers are, however, currently used in clinical applicationsinvolving dental, orthopedic, opthalmological, and surgical uses. Forexample, acrylic-based hydrogels have been used extensively forbioadhesive devices. Acrylic-based hydrogels are well-suited forbioadhesion due to their flexibility and nonabrasive characteristics inthe partially swollen state which reduce damage-causing attrition to thetissues in contact. Park, et al., J. Control. Release 2:47-57, 1985,incorporated herein by reference. Furthermore, their high permeabilityin the swollen state allows unreacted monomer, un-crosslinked polymerchains, and the initiator to be washed out of the matrix afterpolymerization, which is an important feature for selection ofbioadhesive materials for use within the invention. Acrylic-basedpolymer devices exhibit very high adhesive bond strength, as determinedby various known methods. Park, et al., J. Control. Release 2:47-57,1985; Park, et al., Pharm. Res. 4:457-464, 1987; and Ch'ng, et al., J.Pharm. Sci. 74:399-405, 1985, each incorporated herein by reference.

For controlled mucosal delivery of peptide and protein drugs, themethods and compositions of the invention optionally include the use ofcarriers, e.g., polymeric delivery vehicles, that function in part toshield the biologically active agent from proteolytic breakdown, whileat the same time providing for enhanced penetration of the peptide orprotein into or through the nasal mucosa. In this context, bioadhesivepolymers have demonstrated considerable potential for enhancing oraldrug delivery. As an example, the bioavailability of 9-desglycinamide,8-arginine vasopressin (DGAVP) intraduodenally administered to ratstogether with a 1% (w/v) saline dispersion of the mucoadhesivepoly(acrylic acid) derivative polycarbophil, was 3-5-fold increasedcompared to an aqueous solution of the peptide drug without thispolymer. Lehr, et al., J. Pharm. Pharmacol. 44:402-407, 1992,incorporated herein by reference. In this study, the drug was not boundto or otherwise integrally associated with the mucoadhesive polymer inthe formulation, which would therefore not be expected to yield enhancedpeptide absorption via prolonged residence time or intensified contactto the mucosal surface. Thus, certain bioadhesive polymers for usewithin the invention will directly enhance the permeability of theepithelial absorption barrier in part by protecting the active agent,e.g., peptide or protein, from enzymatic degradation.

Recent studies have shown that mucoadhesive polymers of the poly(acrylicacid)-type are potent inhibitors of some intestinal proteases. LueBen,et al., Pharm. Res., 12:1293-1298, 1995; LueBen, et al., J. Control.Rel. 29:329-338, 1994; and Bai, et al., J. Pharm. Sci. 84:1291-1294;1995, incorporated herein by reference. The mechanism of enzymeinhibition is explained by the strong affinity of this class of polymersfor divalent cations, such as calcium or zinc, which are essentialcofactors of metallo-proteinases, such as trypsin and chymotrypsin.Depriving the proteases of their cofactors by poly(acrylic acid) wasreported to induce irreversible structural changes of the enzymeproteins which were accompanied by a loss of enzyme activity. At thesame time, other mucoadhesive polymers (e.g., some cellulose derivativesand chitosan) may not inhibit proteolytic enzymes under certainconditions. In contrast to other enzyme inhibitors contemplated for usewithin the invention (e.g. aprotinin, bestatin), which are relativelysmall molecules, the trans-nasal absorption of inhibitory polymers islikely to be minimal in light of the size of these molecules, andthereby eliminate possible adverse side effects. Thus, mucoadhesivepolymers, particularly of the poly(acrylic acid)-type, may serve both asan absorption-promoting adhesive and enzyme-protective agent to enhancecontrolled delivery of peptide and protein drugs, especially when safetyconcerns are considered.

In addition to protecting against enzymatic degradation, bioadhesivesand other polymeric or non-polymeric absorption-promoting agents for usewithin the invention may directly increase mucosal permeability tobiologically active agents. To facilitate the transport of large andhydrophilic molecules, such as peptides and proteins, across the nasalepithelial barrier, mucoadhesive polymers and other agents have beenpostulated to yield enhanced permeation effects beyond what is accountedfor by prolonged premucosal residence time of the delivery system. Forexample, nasal administration of insulin to non-primate mammals in thepresence of mucoadhesive starch microspheres yielded a steeply enhancedearly absorption peak, followed by a continuous decline. Bjork, et al.,Int. J. Pharm. 47:233-238, 1988; Farraj, et al., J. Control. Rel.13:253-262, 1990, each incorporated herein by reference. The time courseof drug plasma concentrations reportedly suggested that the bioadhesivemicrospheres caused an acute, but transient increase of insulinpermeability across the nasal mucosa. In other studies using in vitrocultured epithelial cell monolayers, Bjork, et al., J. Drug Targeting,1995, incorporated herein by reference, it was reported that dry,swellable materials such as starch microspheres induce reversible focaldilations of the tight junctions, allowing for enhanced drug transportalong the paracellular route. According to this adhesion-dehydrationtheory, the hydrophilic polymer, applied as a dry powder, absorbs waterfrom the mucosal tissue in such a way that the epithelial cells aredehydrated and shrink until the normally tight intercellular junctionsbetween the cells become physically separated. Because this effect is ofrelatively short duration and appears to be completely reversible, itprovides yet another useful tool for incorporation within the coordinateadministration, multi-processing and/or combinatorial formulationmethods and compositions of the invention.

Other mucoadhesive polymers for use within the invention, for examplechitosan, reportedly enhance the permeability of certain mucosalepithelia even when they are applied as an aqueous solution or gel.Lehr, et al., Int. J. Pharmaceut. 78:43-48, 1992; Illum, et al., Pharm.Res. 11:1186-1189, 1994; Artursson, et al., Pharm. Res. 11:1358-1361,1994; and Borchard, et al., J. Control. Release 39:131-138, 1996, eachincorporated herein by reference. In one study, absorption of thepeptide drugs insulin and calcitonin, and the hydrophilic compoundphenol red, from an aqueous gel base of poly(acrylic acid) was reportedafter rectal, vaginal and nasal administration. Morimoto, et al., Int.J. Pharm. 14:149-157, 1983; and Morimoto, et al., J. Pharmacobiodyn10:85-91, 1987, each incorporated herein by reference. Anothermucoadhesive polymer reported to directly affect epithelial permeabilityis hyaluronic acid. In particular, hyaluronic acid gel formulationreportedly enhanced nasal absorption of vasopressin and some of itsanalogues. Morimoto, et al., Pharm. Res. 8:471-474, 1991, incorporatedherein by reference. Hyaluronic acid was also reported to increase theabsorption of insulin from the conjunctiva in diabetic dogs. Nomura, etal., J. Pharm. Pharmacol. 46:768-770, 1994, incorporated herein byreference. Ester derivatives of hyaluronic acid in the form oflyophilized microspheres were described as a nasal delivery system forinsulin. Illum, et al., J. Contr. Rel. 29:133-141, 1994, incorporatedherein by reference.

A particularly useful bioadhesive agent within the coordinateadministration, and/or combinatorial formulation methods andcompositions of the invention is chitosan, as well as its analogs andderivatives. Chitosan is a non-toxic, biocompatible and biodegradablepolymer that is widely used for pharmaceutical and medical applicationsbecause of its favorable properties of low toxicity and goodbiocompatibility. Yomota, Pharm. Tech. Japan 10:557-564, 1994,incorporated herein by reference. It is a natural polyaminosaccharideprepared from chitin by N-deacetylation with alkali. A wide variety ofbiomedical uses for chitosan have been reported over the last twodecades, based for example on its reported wound healing, antimicrobialand hemostatic properties. Kas, J. Microencapsulation 14:689-711, 1997,incorporated herein by reference. Chitosan has also been used as apharmaceutical excipient in conventional dosage forms as well as innovel applications involving bioadhesion and transmucosal drugtransport. Illum, Pharm. Res. 15:1326-1331, 1998; and Olsen, et al.,Chitin and Chitosan-sources, Chemistry, Biochemistry, PhysicalProperties and Applications, 813-828, Skjak-Braek et al., Eds.,Elsevier, London, 1989, each incorporated herein by reference.Furthermore, chitosan has been reported to promote absorption of smallpolar molecules and peptide and protein drugs through nasal mucosa inanimal models and human volunteers. Illum, et al., Pharm. Res.11:1186-1189, 1994, incorporated herein by reference. Other studies haveshown an enhancing effect on penetration of compounds across theintestinal mucosa and cultured Caco-2 cells. Schipper, et al., Pharm.Res. 14:23-29, 1997; and Kotze, et al., Int. J. Pharm. 159:243-253,1997, each incorporated herein by reference. Chitosan has also beenproposed as a bioadhesive polymer for use in oral mucosal drug delivery.Miyazaki, et al., Biol. Pharm. Bull. 17:745-747, 1994; Ikinci, et al.,Advances in Chitin Science, Vol. 4, Peter, et al., Eds., University ofPotsdam, in press; Senel, et al., Int. J. Pharm. 193:197-203, 2000;Needleman, et al., J. Clin. Periodontol. 24:394-400, 1997, eachincorporated herein by reference. Initial studies showed that chitosanhas an extended retention time on the oral mucosa, Needleman, et al., J.Clin. Periodontol. 25:74-82, 1998, incorporated herein by reference, andwith its antimicrobial properties and biocompatibility is an excellentcandidate for the treatment of oral mucositis. More recently, Senel, etal., Biomaterials 21:2067-2071, 2000, incorporated herein by reference,reported that chitosan provides an effective gel carrier for delivery ofthe bioactive peptide, transforming growth factor-β (TGF-β).

As used within the methods and compositions of the invention, chitosanincreases the retention of peptide YY proteins, analogs and mimetics,and other biologically active agents disclosed herein at a mucosal siteof application. This is may be mediated in part by a positive chargecharacteristic of chitosan, which may influence epithelial permeabilityeven after physical removal of chitosan from the surface. Schipper, etal., Pharm. Res. 14:23-29, 1997, incorporated herein by reference.Another mechanism of action of chitosan for improving transport ofbiologically active agents across mucosal membranes may be attributed totransient opening of the tight junctions in the cell membrane to allowpolar compounds to penetrate. Illum, et al., Pharm. Res. 11:1186-1189,1994; Lueben, et al., J. Control. Rel. 29:329-338, 1994, eachincorporated herein by reference. Chitosan may also increase thethermodynamic activity of other absorption-promoting agents used incertain formulations of the invention, resulting in enhancedpenetration. Lastly, as chitosan has been reported to disrupt lipidmicelles in the intestine, Muzzarelli, et al., EUCHIS '99, ThirdInternational Conference of the European Chitin Society, Abstract Book,ORAD-PS-059, Potsdam, Germany, 1999, each incorporated herein byreference, its absorption-promoting effects may be due in part to itsinterference with the lipid organization in the mucosal epithelium.

As with other bioadhesive gels provided herein, the use of chitosan canreduce the frequency of application and the amount of biologicallyactive agent administered while yielding an effective delivery amount ordose. This mode of administration can also improve patient complianceand acceptance. The occlusion and lubrication of chitosan and otherbioadhesive gels is expected to reduce the discomfort of inflammatory,allergic and ulcerative conditions of the nasal mucosa. In addition,chitosan acts non-specifically on certain deleterious microorganisms,including fungi, Knapczyk, Chitin World, pp. 504-511, Karnicki, et al.,Eds., Wirtschaftverlag NR, Germany, 1994, incorporated herein byreference, and may also beneficially stimulate cell proliferation andtissue organization by acting as an inductive primer to repair andphysiologically rebuild damaged tissue. Muzzarelli, et al., Biomaterials10:598-603, 1989, incorporated herein by reference.

As further provided herein, the methods and compositions of theinvention will optionally include a novel chitosan derivative orchemically modified form of chitosan. One such novel derivative for usewithin the invention is denoted as aβ-[1→4]-2-guanidino-2-deoxy-D-glucose polymer (poly-GuD). Chitosan isthe N-deacetylated product of chitin, a naturally occurring polymer thathas been used extensively to prepare microspheres for oral andintra-nasal formulations. The chitosan polymer has also been proposed asa soluble carrier for parenteral drug delivery. Within one aspect of theinvention, o-methylisourea is used to convert a chitosan amine to itsguanidinium moiety. The guanidinium compound is prepared, for example,by the reaction between equi-normal solutions of chitosan ando-methylisourea at pH above 8.0.

The guanidinium product is −[14]-guanidino-2-deoxy-D-glucose polymer. Itis abbreviated as Poly-GuD in this context (Monomer F.W. of Amine inChitosan=161; Monomer F.W. of Guanidinium in Poly-GuD=203).

One exemplary Poly-GuD preparation method for use within the inventioninvolves the following protocol.

Solutions:

Preparation of 0.5% Acetic Acid Solution (0.088N):

-   -   Pipette 2.5 mL glacial acetic acid into a 500 mL volumetric        flask, dilute to volume with purified water.

Preparation of 2N NaOH Solution:

-   -   Transfer about 20 g NaOH pellets into a beaker with about 150 mL        of purified water. Dissolve and cool to room temperature.        Transfer the solution into a 250-mL volumetric flask, dilute to        volume with purified water.

Preparation of O-methylisourea Sulfate (0.4N Urea Group Equivalent):

-   -   Transfer about 493 mg of O-methylisourea sulfate into a 10-mL        volumetric flask, dissolve and dilute to volume with purified        water.    -   The pH of the solution is 4.2

Preparation of Barium Chloride Solution (0.2M):

-   -   Transfer about 2.086 g of Barium chloride into a 50-mL        volumetric flask, dissolve and dilute to volume with purified        water.

Preparation of Chitosan Solution (0.06N Amine Equivalent):

-   -   Transfer about 100 mg Chitosan into a 50 mL beaker, add 10 mL        0.5% Acetic Acid (0.088 N). Stir to dissolve completely.    -   The pH of the solution is about 4.5

Preparation of O-methylisourea Chloride Solution (0.2N Urea GroupEquivalent):

-   -   Pipette 5.0 mL of O-methylisourea sulfate solution (0.4 N urea        group equivalent) and 5 mL of 0.2M Barium chloride solution into        a beaker. A precipitate is formed. Continue to mix the solution        for additional 5 minutes. Filter the solution through 0.45 m        filter and discard the precipitate. The concentration of        O-methylisourea chloride in the supernatant solution is 0.2 N        urea group equivalent.    -   The pH of the solution is 4.2.

Procedure:

-   -   Add 1.5 mL of 2 N NaOH to 10 mL of the chitosan solution (0.06N        amine equivalent) prepared as described in Section 2.5. Adjust        the pH of the solution with 2N NaOH to about 8.2 to 8.4. Stir        the solution for additional 10 minutes. Add 3.0 mL        O-methylisourea chloride solution (0.2N urea group equivalent)        prepared as described above. Stir the solution overnight.    -   Adjust the pH of solution to 5.5 with 0.5% Acetic Acid (0.088N).    -   Dilute the solution to a final volume of 25 mL using purified        water.    -   The Poly-GuD concentration in the solution is 5 mg/mL,        equivalent to 0.025 N (guanidium group).

Additional compounds classified as bioadhesive agents for use within thepresent invention act by mediating specific interactions, typicallyclassified as “receptor-ligand interactions” between complementarystructures of the bioadhesive compound and a component of the mucosalepithelial surface. Many natural examples illustrate this form ofspecific binding bioadhesion, as exemplified by lectin-sugarinteractions. Lectins are (glyco)proteins of non-immune origin whichbind to polysaccharides or glycoconjugates. By virtue of this bindingpotential, lectins may bind or agglutinate cells. Goldstein, et al.,Nature 285:66, 1980, incorporated herein by reference. Lectins arecommonly of plant or bacterial origin, but are also produced by higheranimals (so-called ‘endogenous’ or ‘reverse’ lectins), includingmammals. Sharon, et al., Lectins, Chapman and Hall, London, 1989; andPasztai, et al., Lectins. Biomedical Perspectives, Taylor & Francis,London, 1995, incorporated herein by reference.

Several plant lectins have been investigated as possible pharmaceuticalabsorption-promoting agents. One plant lectin, Phaseolus vulgarishemaglutinin (PHA), exhibits high oral bioavailability of more than 10%after feeding to rats. Pusztai, et al., Biochem. Soc. Trans. 17:81-82,1988, incorporated herein by reference. However, PHA has been reportedto cause digestive disorders following oral administration, and theseside effects must be determined to be minimized by any nasal therapeuticapplication herein. In contrast, tomato (Lycopersicon esculeutum) lectin(TL) appears safe for various modes of administration. This glycoprotein(approximately 70 kDa) resists digestion and binds to rat intestinalvilli without inducing any deleterious effects. Kilpatrick, et al., FEBSLett. 185:5-10, 1985; Woodley, et al., Int. J. Pharm. 110:127-136, 1994;and Int. J. Pharm. 107:223-230, 1994, each incorporated herein byreference. However, GI transit of this radiolabeled lectin afterintragastric administration to rats was not delayed compared tocontrols, and other studies showed that TL has a strong cross-reactivitywith gastrointestinal mucus glycoproteins. Lehr, et al., Pharm. Res.9:547-553, 1992, incorporated herein by reference. Thus, in spite of itsfavorable safety profile, the use of TL as a gastrointestinalbioadhesive, even though its action is “specific” (i.e.,receptor-mediated) is limited by non-specific interactions withmucus—promoting rapid clearance.

Therefore, the invention provides for coordinate administration orcombinatorial formulation of non-toxic lectins identified or obtained bymodification of existing lectins which have a high specific affinity formucosal, e.g., nasal epithelial, cells, but low cross reactivity withmucus. In this regard, detailed teachings regarding lectinstructure-activity relationships will allow selection of non-toxic,strongly bioadhesive candidates to produce optimized lectins fortherapeutic purposes, which undertaking will be further facilitated bymethods of recombinant gene technology. Lehr, et al., Lectins:Biomedical Perspectives, 117-140, Pustai, et al., Eds., Taylor andFrancis, London, 1995, incorporated herein by reference. In additionalembodiments of the invention, mucolytic agents and/or ciliostatic agentsare coordinately administered or combinatorially formulated with abiologically active agent and a lectin or other specific bindingbioadhesive—in order to counter the effects of non-specific binding ofthe bioadhesive to mucosal mucus.

In addition to the use of lectins, certain antibodies or amino acidsequences exhibit high affinity binding to complementary elements oncell and mucosal surfaces. Thus, for example, various adhesive aminoacids sequences such as Arg-Gly-Asp and others, if attached to a carriermatrix, will promote adhesion by binding with specific cell surfaceglycoproteins. In other embodiments, adhesive ligand components areintegrated in a carrier or delivery vehicle that selectively adheres toa particular cell type, or diseased target tissue. For example, certaindiseases cause changes in cell surface glycoproteins. These distinctstructural alterations can be readily targeted by complementary aminoacid sequences bound to a drug delivery vehicle within the invention. Inexemplary aspects, well known cancer-specific markers (e.g., CEA, HER2)may be targeted by complementary antibodies or peptides for specificdrug targeting to diseased cells.

In summary, the foregoing bioadhesive agents are useful in thecombinatorial formulations and coordinate administration methods of theinstant invention, which optionally incorporate an effective amount andform of a bioadhesive agent to prolong persistence or otherwise increasemucosal absorption of one or more peptide YY proteins, analogs andmimetics, and other biologically active agents. The bioadhesive agentsmay be coordinately administered as adjunct compounds or as additiveswithin the combinatorial formulations of the invention. In certainembodiments, the bioadhesive agent acts as a ‘pharmaceutical glue’,whereas in other embodiments adjunct delivery or combinatorialformulation of the bioadhesive agent serves to intensify contact of thebiologically active agent with the nasal mucosa, in some cases bypromoting specific receptor-ligand interactions with epithelial cell“receptors”, and in others by increasing epithelial permeability tosignificantly increase the drug concentration gradient measured at atarget site of delivery (e.g., liver, blood plasma, or CNS tissue orfluid). Yet additional bioadhesive agents for use within the inventionact as enzyme (e.g., protease) inhibitors to enhance the stability ofmucosally administered biotherapeutic agents delivered coordinately orin a combinatorial formulation with the bioadhesive agent.

Liposomes and Micellar Delivery Vehicles

The coordinate administration methods and combinatorial formulations ofthe instant invention optionally incorporate effective lipid or fattyacid based carriers, processing agents, or delivery vehicles, to provideimproved formulations for mucosal delivery of peptide YY proteins,analogs and mimetics, and other biologically active agents. For example,a variety of formulations and methods are provided for mucosal deliverywhich comprise one or more of these active agents, such as a peptide orprotein, admixed or encapsulated by, or coordinately administered with,a liposome, mixed micellar carrier, or emulsion, to enhance chemical andphysical stability and increase the half life of the biologically activeagents (e.g., by reducing susceptibility to proteolysis, chemicalmodification and/or denaturation) upon mucosal delivery.

Within certain aspects of the invention, specialized delivery systemsfor biologically active agents comprise small lipid vesicles known asliposomes. Chonn, et al., Curr. Opin. Biotechnol. 6:698-708, 1995;Lasic, Trends Biotechnol. 16:307-321, 1998; and Gregoriadis, TrendsBiotechnol. 13:527-537, 1995, each incorporated herein by reference.These are typically made from natural, biodegradable, non-toxic, andnon-immunogenic lipid molecules, and can efficiently entrap or bind drugmolecules, including peptides and proteins, into, or onto, theirmembranes. The attractiveness of liposomes as a peptide and proteindelivery system within the invention is increased by the fact that theencapsulated proteins can remain in their preferred aqueous environmentwithin the vesicles, while the liposomal membrane protects them againstproteolysis and other destabilizing factors. Even though not allliposome preparation methods known are feasible in the encapsulation ofpeptides and proteins due to their unique physical and chemicalproperties, several methods allow the encapsulation of thesemacromolecules without substantial deactivation. Weiner, Immunomethods4:201-209, 1994, incorporated herein by reference.

A variety of methods are available for preparing liposomes for usewithin the invention. Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467,1980; and U.S. Pat. Nos. 4,235,871, 4,501,728, and 4,837,028, eachincorporated herein by reference. For use with liposome delivery, thebiologically active agent is typically entrapped within the liposome, orlipid vesicle, or is bound to the outside of the vesicle. Severalstrategies have been devised to increase the effectiveness ofliposome-mediated delivery by targeting liposomes to specific tissuesand specific cell types. Liposome formulations, including thosecontaining a cationic lipid, have been shown to be safe and welltolerated in human patients. Treat, et al., J. Natl. Cancer Instit.82:1706-1710, 1990, incorporated herein by reference.

Like liposomes, unsaturated long chain fatty acids, which also haveenhancing activity for mucosal absorption, can form closed vesicles withbilayer-like structures (so called “ufasomes”). These can be formed, forexample, using oleic acid to entrap biologically active peptides andproteins for mucosal, e.g., intranasal, delivery within the invention.

Other delivery systems for use within the invention combine the use ofpolymers and liposomes to ally the advantageous properties of bothvehicles. Exemplifying this type of hybrid delivery system, liposomescontaining the model protein horseradish peroxidase (HRP) have beeneffectively encapsulated inside the natural polymer fibrin. Henschen, etal., Blood Coagulation, 171-241, Zwaal, et al., Eds., Elsevier,Amsterdam, 1986, incorporated herein by reference. Because of itsbiocompatibility and biodegradability, fibrin is a useful polymer matrixfor drug delivery systems in this context. Senderoff, et al., J.Parenter. Sci. Technol. 45:2-6, 1991; and Jackson, Nat. Med. 2:637-638,1996, incorporated herein by reference. In addition, release ofbiotherapeutic compounds from this delivery system is controllablethrough the use of covalent crosslinking and the addition ofantifibrinolytic agents to the fibrin polymer. Uchino, et al.,Fibrinolysis 5:93-98, 1991, incorporated herein by reference.

More simplified delivery systems for use within the invention includethe use of cationic lipids as delivery vehicles or carriers, which canbe effectively employed to provide an electrostatic interaction betweenthe lipid carrier and such charged biologically active agents asproteins and polyanionic nucleic acids. Hope, et al., Molecular MembraneBiology 15:1-14, 1998, incorporated herein by reference. This allowsefficient packaging of the drugs into a form suitable for mucosaladministration and/or subsequent delivery to systemic compartments.These and related systems are particularly well suited for delivery ofpolymeric nucleic acids, e.g., in the form of gene constructs, antisenseoligonucleotides and ribozymes. These drugs are large, usuallynegatively charged molecules with molecular weights on the order of 10₆for a gene to 10₃ for an oligonucleotide. The targets for these drugsare intracellular, but their physical properties prevent them fromcrossing cell membranes by passive diffusion as with conventional drugs.Furthermore, unprotected DNA is degraded within minutes by nucleasespresent in normal plasma. To avoid inactivation by endogenous nucleases,antisense oligonucleotides and ribozymes can be chemically modified tobe enzyme resistant by a variety of known methods, but plasmid DNA mustordinarily be protected by encapsulation in viral or non-viralenvelopes, or condensation into a tightly packed particulate form bypolycations such as proteins or cationic lipid vesicles. More recently,small unilamellar vesicles (SUVs) composed of a cationic lipid anddioleoylphosphatidylethanolamine (DOPE) have been successfully employedas vehicles for polynucleic acids, such as plasmid DNA, to formparticles capable of transportation of the active polynucleotide acrossplasma membranes into the cytoplasm of a broad spectrum of cells. Thisprocess (referred to as lipofection or cytofection) is now widelyemployed as a means of introducing plasmid constructs into cells tostudy the effects of transient gene expression. Exemplary deliveryvehicles of this type for use within the invention include cationiclipids (e.g., N-(2,3-(dioleyloxy)propyl)-N,N,N-trimethyl ammoniumchloride (DOTMA)), quarternary ammonium salts (e.g.,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC)), cationic derivativesof cholesterol (e.g., 3β(N-(N′,N-dimethylaminoethane-carbamoyl-cholesterol (DC-chol)), and lipidscharacterized by multivalent headgroups (e.g.,dioctadecyldimethylammonium chloride (DOGS), commercially available asTransfectam®).

Additional delivery vehicles for use within the invention include longand medium chain fatty acids, as well as surfactant mixed micelles withfatty acids. Muranishi, Crit. Rev. Ther. Drug Carrier Syst. 7:1-33,1990, incorporated herein by reference. Most naturally occurring lipidsin the form of esters have important implications with regard to theirown transport across mucosal surfaces. Free fatty acids and theirmonoglycerides which have polar groups attached have been demonstratedin the form of mixed micelles to act on the intestinal barrier aspenetration enhancers. This discovery of barrier modifying function offree fatty acids (carboxylic acids with a chain length varying from 12to 20 carbon atoms) and their polar derivatives has stimulated extensiveresearch on the application of these agents as mucosal absorptionenhancers.

For use within the methods of the invention, long chain fatty acids,especially fusogenic lipids (unsaturated fatty acids and monoglyceridessuch as oleic acid, linoleic acid, linoleic acid, monoolein, etc.)provide useful carriers to enhance mucosal delivery of peptide YYproteins, analogs and mimetics, and other biologically active agentsdisclosed herein. Medium chain fatty acids (C6 to C12) andmonoglycerides have also been shown to have enhancing activity inintestinal drug absorption and can be adapted for use within the mocosaldelivery formulations and methods of the invention. In addition, sodiumsalts of medium and long chain fatty acids are effective deliveryvehicles and absorption-enhancing agents for mucosal delivery ofbiologically active agents within the invention. Thus, fatty acids canbe employed in soluble forms of sodium salts or by the addition ofnon-toxic surfactants, e.g., polyoxyethylated hydrogenated castor oil,sodium taurocholate, etc. Mixed micelles of naturally occurringunsaturated long chain fatty acids (oleic acid or linoleic acid) andtheir monoglycerides with bile salts have been shown to exhibitabsorption-enhancing abilities which are basically harmless to theintestinal mucosa. Muranishi, Pharm. Res. 2:108-118, 1985; and Crit.Rev. Ther. Drug Carrier Syst. 7:1-33, 1990, each incorporated herein byreference. Other fatty acid and mixed micellar preparations that areuseful within the invention include, but are not limited to, Nacaprylate (C8), Na caprate (C10), Na laurate (C12) or Na oleate (C18),optionally combined with bile salts, such as glycocholate andtaurocholate.

Pegylation

Additional methods and compositions provided within the inventioninvolve chemical modification of biologically active peptides andproteins by covalent attachment of polymeric materials, for exampledextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol andpolyamino acids. The resulting conjugated peptides and proteins retaintheir biological activities and solubility for mucosal administration.In alternate embodiments, peptide YY proteins, analogs and mimetics, andother biologically active peptides and proteins, are conjugated topolyalkylene oxide polymers, particularly polyethylene glycols (PEG).U.S. Pat. No. 4,179,337, incorporated herein by reference. Numerousreports in the literature describe the potential advantages of pegylatedpeptides and proteins, which often exhibit increased resistance toproteolytic degradation, increased plasma half-life, increasedsolubility and decreased antigenicity and immunogenicity. Nucci, et al.,Advanced Drug Deliver Reviews 6:133-155, 1991; Lu, et al., Int. J.Peptide Protein Res. 43:127-138, 1994, each incorporated herein byreference. A number of proteins, including L-asparaginase,strepto-kinase, insulin, interleukin-2, adenosine deamidase,L-asparaginase, interferon alpha 2b, superoxide dismutase,streptokinase, tissue plasminogen activator (tPA), urokinase, uricase,hemoglobin, TGF-beta, EGF, and other growth factors, have beenconjugated to PEG and evaluated for their altered biochemical propertiesas therapeutics. Ho, et al., Drug Metabolism and Disposition 14:349-352,1986; Abuchowski, et al., Prep. Biochem. 9:205-211, 1979; andRajagopaian, et al., J. Clin. Invest. 75:413-419, 1985, Nucci, et al.,Adv. Drug Delivery Rev. 4:133-151, 1991, each incorporated herein byreference. Although the in vitro biological activities of pegylatedproteins may be decreased, this loss in activity is usually offset bythe increased in vivo half-life in the bloodstream. Nucci, et al.,Advanced Drug Deliver Reviews 6:133-155, 1991, incorporated herein byreference. Accordingly, these and other polymer-coupled peptides andproteins exhibit enhanced properties, such as extended half-life andreduced immunogenicity, when administered mucoally according to themethods and formulations herein.

Several procedures have been reported for the attachment of PEG toproteins and peptides and their subsequent purification. Abuchowski, etal., J. Biol. Chem. 252:3582-3586, 1977; Beauchamp, et al., Anal.Biochem. 131:25-33, 1983, each incorporated herein by reference. Inaddition, Lu, et al., Int. J. Peptide Protein Res. 43:127-138, 1994,incorporated herein by reference, describe various technicalconsiderations and compare PEGylation procedures for proteins versuspeptides. Katre, et al., Proc. Natl. Acad. Sci. U.S.A. 84:1487-1491,1987; Becker, et al., Makromol. Chem. Rapid Commun. 3:217-223, 1982;Mutter, et al., Makromol. Chem. Rapid Commun. 13:151-157, 1992;Merrifield, R. B., J. Am. Chem. Soc. 85:2149-2154, 1993; Lu, et al.,Peptide Res. 6:142-146, 1993; Lee, et al., Bioconjugate Chem.10:973-981, 1999, Nucci, et al., Adv. Drug Deliv. Rev. 6:133-151, 1991;Francis, et al., J. Drug Targeting 3:321-340, 1996; Zalipsky, S.,Bioconjugate Chem. 6:150-165, 1995; Clark, et al., J. Biol. Chem.271:21969-21977, 1996; Pettit, et al., J. Biol. Chem., 272:2312-2318,1997; Delgado, et al., Br. J. Cancer 73:175-182, 1996; Benhar, et al.,Bioconjugate Chem. 5:321-326, 1994; Benhar, et al., J. Biol. Chem.269:13398-13404, 1994; Wang, et al., Cancer Res. 53:4588-4594, 1993;Kinstler, et al., Pharm. Res. 13:996-1002, 1996, Filpula, et al., Exp.Opin. Ther. Patents 9:231-245, 1999; Pelegrin, et al., Hum. Gene Ther.9:2165-2175, 1998, each incorporated herein by reference.

Following these and other teachings in the art, the conjugation ofbiologically active peptides and proteins for with polyethyleneglycolpolymers, is readily undertaken, with the expected result of prolongingcirculating life and/or reducing immunogenicity while maintaining anacceptable level of activity of the PEGylated active agent.Amine-reactive PEG polymers for use within the invention include SC-PEGwith molecular masses of 2000, 5000, 10000, 12000, and 20 000;U-PEG-10000; NHS-PEG-3400-biotin; T-PEG-5000; T-PEG-12000; andTPC-PEG-5000. Chemical conjugation chemistries for these polymers havebeen published. Zalipsky, S., Bioconjugate Chem. 6:150-165, 1995;Greenwald, et al., Bioconjugate Chem. 7:638-641, 1996; Martinez, et al.,Macromol. Chem. Phys. 198:2489-2498, 1997; Hermanson, G. T.,Bioconjugate Techniques, 605-618, 1996; Whitlow, et al., Protein Eng.6:989-995, 1993; Habeeb, A. F. S. A., Anal. Biochem. 14:328-336, 1966;Zalipsky, et al., Poly(ethyleneglycol) Chemistry and BiologicalApplications, 318-341, 1997; Harlow, et al., Antibodies: A LaboratoryManual, 553-612, Cold Spring Harbor Laboratory, Plainview, N.Y., 1988;Milenic, et al., Cancer Res. 51:6363-6371, 1991; Friguet, et al., J.Immunol. Methods 77:305-319, 1985, each incorporated herein byreference. While phosphate buffers are commonly employed in theseprotocols, the choice of borate buffers may beneficially influence thePEGylation reaction rates and resulting products.

PEGylation of biologically active peptides and proteins may be achievedby modification of carboxyl sites (e.g., aspartic acid or glutamic acidgroups in addition to the carboxyl terminus). The utility ofPEG-hydrazide in selective modification of carbodiimide-activatedprotein carboxyl groups under acidic conditions has been described.Zalipsky, S., Bioconjugate Chem. 6:150-165, 1995; Zalipsky, et al.,Poly(ethyleneglycol) Chemistry and Biological Applications, 318-341,American Chemical Society, Washington, D.C., 1997, incorporated hereinby reference. Alternatively, bifunctional PEG modification ofbiologically active peptides and proteins can be employed. In someprocedures, charged amino acid residues, including lysine, asparticacid, and glutamic acid, have a marked tendency to be solvent accessibleon protein surfaces. Conjugation to carboxylic acid groups of proteinsis a less frequently explored approach for production of proteinbioconjugates. However, the hydrazide/EDC chemistry described byZalipsky and colleagues, Zalipsky, S., Bioconjugate Chem. 6:150-165,1995; Zalipsky, et al., Poly(ethyleneglycol) Chemistry and BiologicalApplications, 318-341, American Chemical Society, Washington, D.C.,1997, each incorporated herein by reference, offers a practical methodof linking PEG polymers to protein carboxylic sites. For example, thisalternate conjugation chemistry has been shown to be superior to aminelinkages for PEGylation of brain-derived neurotrophic factor (BDNF)while retaining biological activity. Wu, et al., Proc. Natl. Acad. Sci.U.S.A. 96:254-259, 1999, incorporated herein by reference. Maeda andcolleagues have also found carboxyl-targeted PEGylation to be thepreferred approach for bilirubin oxidase conjugations. Maeda, et al.,Poly(ethylene glycol) Chemistry. Biotechnical and BiomedicalApplications, J. M. Harris, Ed., pp. 153-169, Plenum Press, New York,1992, incorporated herein by reference.

Often, PEGylation of peptides and proteins for use within the inventioninvolves activating PEG with a functional group that will react withlysine residues on the surface of the peptide or protein. Within certainalternate aspects of the invention, biologically active peptides andproteins are modified by PEGylation of other residues such as His, Trp,Cys, Asp, Glu, etc., without substantial loss of activity. If PEGmodification of a selected peptide or protein proceeds to completion,the activity of the peptide or protein is often diminished. Therefore,PEG modification procedures herein are generally limited to partialPEGylation of the peptide or protein, resulting in less than about 50%,more commonly less than about 25%, loss of activity, while providing forsubstantially increased half-life (e.g., serum half life) and asubstantially decreased effective dose requirement of the PEGylatedactive agent.

An unavoidable result of partial PEG modification is the production of aheterogenous mixture of PEGylated peptide or protein having astatistical distribution of the number of PEG groups bound per molecule.In addition, the usage of lysine residues within the peptide or proteinis random. These two factors result in the production of a heterogeneousmixture of PEGylated proteins which differ in both the number andposition of the PEG groups attached. For instance, when adenosinedeaminase is optimally modified there is a loss of 50% activity when theprotein has about 14 PEG per protein, with a broad distribution of theactual number of PEG moieties per individual protein and a broaddistribution of the position of the actual lysine residues used. Suchmixtures of diversely modified proteins are not optimally suited forpharmaceutical use. At the same time, purification and isolation of aclass of PEGylated proteins (e.g., proteins containing the same numberof PEG moieties) or a single type of PEGylated protein (e.g., proteinscontaining both the same number of moieties and having the PEG moietiesat the same position) involves time-consuming and expensive procedureswhich result in an overall reduction in the yield of the specificPEGylated peptide or protein of interest.

Within certain alternate aspects of the invention, biologically activepeptides and proteins are modified by PEGylation methods that employactivated PEG reagents that react with thio groups of the protein,resulting in covalent attachment of PEG to a cysteine residue, whichresidue may be inserted in place of a naturally-occurring lysine residueof the protein. As described, for example, in U.S. Pat. No. 5,166,322(incorporated herein by reference) specific variants of IL-3 have beensuccessfully produced which have a cysteine residue introduced atselected sites within the naturally occurring amino acid sequence.Sulfhydryl reactive compounds (e.g. activated polyethylene glycol) arethen attached to these cysteines by reaction with the IL-3 variant.Additionally, U.S. Pat. No. 5,206,344 (incorporated herein by reference)describes specific IL-2 variants which contain a cysteine residueintroduced at selected sites within the naturally-occurring amino acidsequence. The IL-2 variant is subsequently reacted with an activatedpolyethylene glycol reagent to attach this moiety to a cysteine residue.

Yet additional methods employed within the invention for generatingPEGylated peptides and proteins do not require extensive knowledge ofprotein structure-function (e.g., mapping amino acid residues essentialfor biological activity). Exemplifying these methods, U.S. Pat. No.5,766,897, incorporated herein by reference, describes methods forproduction and characterization of cysteine-PEGylated proteins suitablefor therapeutic applications. These are produced by attaching apolyethylene glycol to a cysteine residue within the protein. To obtainthe desired result of a stable, biologically active compound the PEG isattached in a specific manner, often to a cysteine residue present at ornear a site that is normally glycosylated. Typically, the specific aminoacid modified by glycosylation (e.g., asparagine in N-linkedglycosylation or serine or threonine in O-linked glycosylation) isreplaced by a cysteine residue, which is subsequently chemicallymodified by attachment of PEG. It may be useful for employment of thismethod to generation cysteine-containing mutants of selectedbiologically active peptides and proteins, which can be readilyaccomplished by, for example, site-directed mutagenesis using methodswell known in the art. Kunkel, in Nucleic Acids and Molecular Biology,Eckstein, F. Lilley, D. M. J., eds., Springer-Verlag, Berling andHeidelberg, vol. 2, p. 124, 1988, incorporated herein by reference. Inaddition, if the active peptide or protein is one member of a family ofstructurally related proteins, glycosylation sites for any other membercan be matched to an amino acid on the protein of interest, and thatamino acid changed to cysteine for attachment of the polyethyleneglycol. Alternatively, if a crystal structure has been determined forthe protein of interest or a related protein, surface residues away fromthe active site or binding site can be changed to cysteine for theattachment of polyethylene glycol.

These strategies for identifying useful PEG attachment sites for usewithin the invention are advantageous in that they are readilyimplemented without extensive knowledge of protein structure-functiondetails. Moreover, these strategies also take advantage of the fact thatthe presence and location of glycosylation residues are often related,as a natural evolutionary consequence, to increased stability and serumhalf-life of the subject peptide or protein. Replacement of theseglycosylation residues by cysteine, followed by cysteine-specificPEGylation, commonly yields modified peptides and proteins that retainsubstantial biological activity while exhibiting significantly increasedstability.

If a higher degree of PEG modification is required, and/or if thepeptide or protein to be chemically modified is not normallyglycosylated, other solvent accessible residues can be changed tocysteine, and the resultant protein subjected to PEGylation. Appropriateresidues can easily be determined by those skilled in the art. Forinstance, if a three-dimensional structure is available for the proteinof interest, or a related protein, solvent accessible amino acids areeasily identified. Also, charged amino acids such as Lys, Arg, Asp andGlu are almost exclusively found on the surface of proteins.Substitution of one, two or many of these residues with cysteine willprovide additional sites for PEG attachment. In addition, amino acidsequences in the native protein that are recognized by antibodies areusually on the surface of the protein. These and other methods fordetermining solvent accessible amino acids are well known to thoseskilled in the art.

Modification of peptides and proteins with PEG can also be used togenerate multimeric complexes of proteins, fragments, and/or peptidesthat have increased biological stability and/or potency. Thesemultimeric peptides and proteins of the invention, e.g., dimers ortetramers of peptide YY, may be produced synthetically according to wellknown methods. Alternatively, other biologically active peptides andproteins may be produced in this manner that are naturally occurringdimeric or multimeric proteins. For example, dimeric peptides andproteins useful within the invention may be produced by reacting thepeptide or protein with (Maleimido)₂-PEG, a reagent composed of PEGhaving two protein-reactive moieties. In the case of cysteine-pegylatedpeptides and proteins, the degree of multimeric cross-linking can becontrolled by the number of cysteines either present and/or engineeredinto the peptide or protein, and by the concentration of reagents, e.g.,(Maleimido)₂ PEG, used in the reaction mixture.

It is further contemplated to attach other groups to thio groups ofcysteines present in biologically active peptides and proteins for usewithin the invention. For example, the peptide or protein may bebiotinylated by attaching biotin to a thio group of a cysteine residue.Examples of cysteine-PEGylated proteins of the invention, as well asproteins having a group other than PEG covalently attached via acysteine residue according to the invention, are as follows:

Other Stabilizing Modifications of Active Agents

In addition to PEGylation, biologically active agents such as peptidesand proteins for use within the invention can be modified to enhancecirculating half-life by shielding the active agent via conjugation toother known protecting or stabilizing compounds, for example by thecreation of fusion proteins with an active peptide, protein, analog ormimetic linked to one or more carrier proteins, such as one or moreimmunoglobulin chains. U.S. Pat. Nos. 5,750,375; 5,843,725; 5,567,584and 6,018,026, each incorporated herein by reference. Thesemodifications will decrease the degradation, sequestration or clearanceof the active agent and result in a longer half-life in a physiologicalenvironment (e.g., in the circulatory system, or at a mucosal surface).The active agents modified by these and other stabilizing conjugationsmethods are therefore useful with enhanced efficacy within the methodsof the invention. In particular, the active agents thus modifiedmaintain activity for greater periods at a target site of delivery oraction compared to the unmodified active agent. Even when the activeagent is thus modified, it retains substantial biological activity incomparison to a biological activity of the unmodified compound.

Thus, in certain aspects of the invention, peptide YY proteins, analogsand mimetics, and other biologically active agents, including otheractive peptides and proteins, for mucosal administration according tothe methods of the invention are modified for enhanced activity, e.g.,to increase circulating half-life, by shielding the active agent throughconjugation to other known protecting or stabilizing compounds, or bythe creation of fusion proteins with the peptide, protein, analog ormimetic linked to one or more carrier proteins, such as one or moreimmunoglobulin chains. U.S. Pat. Nos. 5,750,375; 5,843,725; 5,567,584;and 6,018,026, each incorporated herein by reference. Thesemodifications will decrease the degradation, sequestration or clearanceof the active peptide or protein and result in a longer half-life in aphysiological environment (e.g., at the nasal mucosal surface or in thesystemic circulation). The active peptides and proteins thus modifiedexhibit enhanced efficacy within the compositions and methods of theinvention, for example by increased or temporally extended activity at atarget site of delivery or action compared to the unmodified peptide,protein, analog or mimetic.

In other aspects of the invention, peptide and protein therapeuticcompounds are conjugated for enhanced stability with relatively lowmolecular weight compounds, such as aminolethicin, fatty acids, vitaminB₁₂, and glycosides. Igarishi, et al., Proc. Int. Symp. Control. Rel.Bioact. Materials 17:366, 1990, incorporated herein by reference.Additional exemplary modified peptides and proteins for use within thecompositions and methods of the invention will be beneficially modifiedfor in vivo use by:

(a) chemical or recombinant DNA methods to link mammalian signalpeptides, Lin, et al., J. Biol. Chem. 270:14255, 1995, incorporatedherein by reference, or bacterial peptides, Joliot, et al., Proc. Natl.Acad. Sci. U.S.A. 88:1864, 1991, incorporated herein by reference, tothe active peptide or protein, which serves to direct the active peptideor protein across cytoplasmic and organellar membranes and/or trafficthe active peptide or protein to the a desired intracellular compartment(e.g., the endoplasmic reticulum (ER) of antigen presenting cells(APCs), such as dendritic cells for enhanced CTL induction);

(b) addition of a biotin residue to the active peptide or protein whichserves to direct the active conjugate across cell membranes by virtue ofits ability to bind specifically (i.e., with a binding affinity greaterthan about 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹) to a translocator present onthe surface of cells (Chen, et al., Analytical Biochem. 227:168, 1995,incorporated herein by reference);

(c) addition at either or both the amino- and carboxy-terminal ends ofthe active peptide or protein of a blocking agent in order to increasestability in vivo. This can be useful in situations in which the terminiof the active peptide or protein tend to be degraded by proteases priorto cellular uptake or during intracellular trafficking. Such blockingagents can include, without limitation, additional related or unrelatedpeptide sequences that can be attached to the amino and/or carboxyterminal residues of the therapeutic polypeptide or peptide to beadministered. This can be done either chemically during the synthesis ofthe peptide or by recombinant DNA technology. Blocking agents such aspyroglutamic acid or other molecules known to those skilled in the artcan also be attached to the amino and/or carboxy terminal residues, orthe amino group at the amino terminus or carboxyl group at the carboxyterminus can be replaced with a different moiety.

Biologically active agents modified by PEGylation and other stabilizingmethods for use within the methods and compositions of the inventionwill preferably retain at least 25%, more preferably at least 50%, evenmore preferably between about 50% to 75%, most preferably 100% of thebiological activity associated with the unmodified active agent, e.g., anative peptide or protein. Typically, the modified active agent, e.g., aconjugated peptide or protein, has a half-life (t_(1/2)), for example inserum following mucosal delivery, which is enhanced relative to thehalf-life of the unmodified active agent from which it was derived. Incertain aspects, the half-life of a modified active agent (e.g., peptideYY proteins, analogs and mimetics, and other biologically activepeptides and proteins disclosed herein) for use within the invention isenhanced by at least 1.5-fold to 2-fold, often by about 2-fold to3-fold, in other cases by about 5-fold to 10-fold, and up to 100-fold ormore relative to the half-life of the unmodified active agent.

Prodrug Modifications

Yet another processing and formulation strategy useful within theinvention is that of prodrug modification. By transiently (i.e.,bioreversibly) derivatizing such groups as carboxyl, hydroxyl, and aminogroups in small organic molecules, the undesirable physicochemicalcharacteristics (e.g., charge, hydrogen bonding potential, etc. thatdiminish mucosal penetration) of these molecules can be “masked” withoutpermanently altering the pharmacological properties of the molecule.Bioreversible prodrug derivatives of therapeutic small molecule drugshave been shown to improve the physicochemical (e.g., solubility,lipophilicity) properties of numerous exemplary therapeutics,particularly those that contain hydroxyl and carboxylic acid groups.

One approach to making prodrugs of amine-containing active agents, suchas the peptides and proteins of the invention, is through the acylationof the amino group. Optionally, the use of acyloxyalkoxycarbamatederivatives of amines as prodrugs has been discussed.3-(2′-hydroxy-4′,6′-dimethylphenyl)-3,3-dimethylpropionic acid has beenemployed to prepare linear, esterase-, phosphatase-, anddehydrogenase-sensitive prodrugs of amines (Amsberry, et al., Pharm.Res. 8:455-461, 1991; Wolfe, et al., J. Org. Chem. 57:6138, 1992, eachincorporated herein by reference). These systems have been shown todegrade through a two-step mechanism, with the first step being theslow, rate-determining enzyme-catalyzed (esterase, phosphatase, ordehydrogenase) step, and the second step being a rapid (t_(1/2)=100sec., pH 7.4, 37° C.) chemical step (Amsberry, et al., J. Org. Chem.55:5867-5877, 1990, incorporated herein by reference). Interestingly,the phosphatase-sensitive system has recently been employed to prepare avery water-soluble (greater than 10 mg/ml) prodrug of TAXOL which showssignificant antitumor activity in vivo. These and other prodrugmodification systems and resultant therapeutic agents are useful withinthe methods and compositions of the invention.

For the purpose of preparing prodrugs of peptides that are useful withinthe invention, U.S. Pat. No. 5,672,584 (incorporated herein byreference) further describes the preparation and use of cyclic prodrugsof biologically active peptides and peptide nucleic acids (PNAs). Toproduce these cyclic prodrugs, the N-terminal amino group and theC-terminal carboxyl group of a biologically active peptide or PNA islinked via a linker, or the C-terminal carboxyl group of the peptide islinked to a side chain amino group or a side chain hydroxyl group via alinker, or the N-terminal amino group of the peptide is linked to a sidechain carboxyl group via a linker, or a side chain carboxyl group of thepeptide is linked to a side chain amino group or a side chain hydroxylgroup via a linker. Useful linkers in this context include3-(2′-hydroxy-4′,6′-dimethyl phenyl)-3,3-dimethyl propionic acid linkersand its derivatives, and acyloxyalkoxy derivatives. The incorporateddisclosure provides methods useful for the production andcharacterization of cyclic prodrugs synthesized from linear peptides,e.g., opioid peptides that exhibit advantageous physicochemical features(e.g., reduced size, intramolecular hydrogen bond, and amphophiliccharacteristics) for enhanced cell membrane permeability and metabolicstability. These methods for peptide prodrug modification are alsouseful to prepare modified peptide therapeutic derivatives for usewithin the methods and compositions of the invention.

Purification and Preparation

Biologically active agents for mucosal administration according to theinvention, for example peptide YY proteins, analogs and mimetics, andother biologically active agents disclosed herein, are generallyprovided for direct administration to subjects in a substantiallypurified form. The term “substantially purified” as used herein, isintended to refer to a peptide, protein, nucleic acid or other compoundthat is isolated in whole or in part from naturally associated proteinsand other contaminants, wherein the peptide, protein, nucleic acid orother active compound is purified to a measurable degree relative to itsnaturally-occurring state, e.g., relative to its purity within a cellextract.

In certain embodiments, the term “substantially purified” refers to apeptide, protein, or polynucleotide composition that has been isolatedfrom a cell, cell culture medium, or other crude preparation andsubjected to fractionation to remove various components of the initialpreparation, such as proteins, cellular debris, and other components. Ofcourse, such purified preparations may include materials in covalentassociation with the active agent, such as glycoside residues ormaterials admixed or conjugated with the active agent, which may bedesired to yield a modified derivative or analog of the active agent orproduce a combinatorial therapeutic formulation, conjugate, fusionprotein or the like. The term purified thus includes such desiredproducts as peptide and protein analogs or mimetics or otherbiologically active compounds wherein additional compounds or moietiessuch as polyethylene glycol, biotin or other moieties are bound to theactive agent in order to allow for the attachment of other compoundsand/or provide for formulations useful in therapeutic treatment ordiagnostic procedures.

As applied to polynucleotides, the term substantially purified denotesthat the polynucleotide is free of substances normally accompanying it,but may include additional sequence at the 5′ and/or 3′ end of thecoding sequence which might result, for example, from reversetranscription of the noncoding portions of a message when the DNA isderived from a cDNA library, or might include the reverse transcript forthe signal sequence as well as the mature protein encoding sequence.

When referring to peptides, proteins and peptide analogs (includingpeptide fusions with other peptides and/or proteins) of the invention,the term substantially purified typically means a composition which ispartially to completely free of other cellular components with which thepeptides, proteins or analogs are associated in a non-purified, e.g.,native state or environment. Purified peptides and proteins aregenerally in a homogeneous or nearly homogenous state although it can beeither in a dry state or in an aqueous solution. Purity and homogeneityare typically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography.

Generally, substantially purified peptides, proteins and other activecompounds for use within the invention comprise more than 80% of allmacromolecular species present in a preparation prior to admixture orformulation of the peptide, protein or other active agent with apharmaceutical carrier, excipient, buffer, absorption enhancing agent,stabilizer, preservative, adjuvant or other co-ingredient in a completepharmaceutical formulation for therapeutic administration. Moretypically, the peptide or other active agent is purified to representgreater than 90%, often greater than 95% of all macromolecular speciespresent in a purified preparation prior to admixture with otherformulation ingredients. In other cases, the purified preparation ofactive agent may be essentially homogeneous, wherein othermacromolecular species are not detectable by conventional techniques.

Various techniques suitable for use in peptide and protein purificationare well known to those of skill in the art. These include, for example,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite and/oraffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. Particularly usefulpurification methods include selective precipitation with suchsubstances as ammonium sulfate; column chromatography; affinity methods,including immunopurification methods; and others. Scopes, R., ProteinPurification: Principles and Practice, Springer-Verlag: New York, 1982,incorporated herein by reference. In general, biologically activepeptides and proteins can be extracted from tissues or cell culturesthat express the peptides and then immunoprecipitated, where after thepeptides and proteins can be further purified by standard proteinchemistry/chromatographic methods.

Formulation and Administration

Mucosal delivery formulations of the present invention comprise thebiologically active agent to be administered (e.g., one or more of thepeptide YY proteins, analogs and mimetics, and other biologically activeagents disclosed herein), typically combined together with one or morepharmaceutically acceptable carriers and, optionally, other therapeuticingredients. The carrier(s) must be “pharmaceutically acceptable” in thesense of being compatible with the other ingredients of the formulationand not eliciting an unacceptable deleterious effect in the subject.Such carriers are described herein above or are otherwise well known tothose skilled in the art of pharmacology. Desirably, the formulationshould not include substances such as enzymes or oxidizing agents withwhich the biologically active agent to be administered is known to beincompatible. The formulations may be prepared by any of the methodswell known in the art of pharmacy.

Within the compositions and methods of the invention, the peptide YYproteins, analogs and mimetics, and other biologically active agentsdisclosed herein may be administered to subjects by a variety of mucosaladministration modes, including by oral, rectal, vaginal, intranasal,intrapulmonary, or transdermal delivery, or by topical delivery to theeyes, ears, skin or other mucosal surfaces. Optionally, peptide YYproteins, analogs and mimetics, and other biologically active agentsdisclosed herein can be coordinately or adjunctively administered bynon-mucosal routes, including by intramuscular, subcutaneous,intravenous, intra-atrial, intra-articular, intraperitoneal, orparenteral routes. In other alternative embodiments, the biologicallyactive agent(s) can be administered ex vivo by direct exposure to cells,tissues or organs originating from a mammalian subject, for example as acomponent of an ex vivo tissue or organ treatment formulation thatcontains the biologically active agent in a suitable, liquid or solidcarrier.

Compositions according to the present invention are often administeredin an aqueous solution as a nasal or pulmonary spray and may bedispensed in spray form by a variety of methods known to those skilledin the art. Preferred systems for dispensing liquids as a nasal sprayare disclosed in U.S. Pat. No. 4,511,069. Such formulations may beconveniently prepared by dissolving compositions according to thepresent invention in water to produce an aqueous solution, and renderingthe solution sterile. The formulations may be presented in multi-dosecontainers, for example in the sealed dispensing system disclosed inU.S. Pat. No. 4,511,069. Other suitable nasal spray delivery systemshave been described in Transdermal Systemic Medication, Chien, Y. W.Ed., Elsevier Publishers, New York, 1985; and in U.S. Pat. No. 4,778,810(each incorporated herein by reference). Additional aerosol deliveryforms may include, e.g., compressed air-, jet-, ultrasonic-, andpiezoelectric nebulizers, which deliver the biologically active agentdissolved or suspended in a pharmaceutical solvent, e.g., water,ethanol, or a mixture thereof.

Nasal and pulmonary spray solutions of the present invention typicallycomprise the drug or drug to be delivered, optionally formulated with asurface active agent, such as a nonionic surfactant (e.g.,polysorbate-80), and one or more buffers. In some embodiments of thepresent invention, the nasal spray solution further comprises apropellant. The pH of the nasal spray solution is optionally betweenabout pH 6.8 and 7.2, but when desired the pH is adjusted to optimizedelivery of a charged macromolecular species (e.g., a therapeuticprotein or peptide) in a substantially unionized state. Thepharmaceutical solvents employed can also be a slightly acidic aqueousbuffer (pH 4-6). Suitable buffers for use within these compositions areas described above or as otherwise known in the art. Other componentsmay be added to enhance or maintain chemical stability, includingpreservatives, surfactants, dispersants, or gases. Suitablepreservatives include, but are not limited to, phenol, methyl paraben,paraben, m-cresol, thiomersal, benzylalkonium chloride, and the like.Suitable surfactants include, but are not limited to, oleic acid,sorbitan trioleate, polysorbates, lecithin, phosphotidyl cholines, andvarious long chain diglycerides and phospholipids. Suitable dispersantsinclude, but are not limited to, ethylenediaminetetraacetic acid, andthe like. Suitable gases include, but are not limited to, nitrogen,helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbondioxide, air, and the like.

Within alternate embodiments, mucosal formulations are administered asdry powder formulations comprising the biologically active agent in adry, usually lyophilized, form of an appropriate particle size, orwithin an appropriate particle size range, for intranasal delivery.Minimum particle size appropriate for deposition within the nasal orpulmonary passages is often about 0.5μ mass median equivalentaerodynamic diameter (MMEAD), commonly about 1μ MMEAD, and moretypically about 2μ MMEAD. Maximum particle size appropriate fordeposition within the nasal passages is often about 10μ MMEAD, commonlyabout 8μ MMEAD, and more typically about 4μ MMEAD. Intranasallyrespirable powders within these size ranges can be produced by a varietyof conventional techniques, such as jet milling, spray drying, solventprecipitation, supercritical fluid condensation, and the like. These drypowders of appropriate MMEAD can be administered to a patient via aconventional dry powder inhaler (DPI) which rely on the patient'sbreath, upon pulmonary or nasal inhalation, to disperse the power intoan aerosolized amount. Alternatively, the dry powder may be administeredvia air assisted devices that use an external power source to dispersethe powder into an aerosolized amount, e.g., a piston pump.

Dry powder devices typically require a powder mass in the range fromabout 1 mg to 20 mg to produce a single aerosolized dose (“puff”). Ifthe required or desired dose of the biologically active agent is lowerthan this amount, the powdered active agent will typically be combinedwith a pharmaceutical dry bulking powder to provide the required totalpowder mass. Preferred dry bulking powders include sucrose, lactose,dextrose, mannitol, glycine, trehalose, human serum albumin (HSA), andstarch. Other suitable dry bulking powders include cellobiose, dextrans,maltotriose, pectin, sodium citrate, sodium ascorbate, and the like.

To formulate compositions for mucosal delivery within the presentinvention, the biologically active agent can be combined with variouspharmaceutically acceptable additives, as well as a base or carrier fordispersion of the active agent(s). Desired additives include, but arenot limited to, pH control agents, such as arginine, sodium hydroxide,glycine, hydrochloric acid, citric acid, etc. In addition, localanesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodiumchloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80),solubility enhancing agents (e.g., cyclodextrins and derivativesthereof), stabilizers (e.g., serum albumin), and reducing agents (e.g.,glutathione) can be included. When the composition for mucosal deliveryis a liquid, the tonicity of the formulation, as measured with referenceto the tonicity of 0.9% (w/v) physiological saline solution taken asunity, is typically adjusted to a value at which no substantial,irreversible tissue damage will be induced in the nasal mucosa at thesite of administration. Generally, the tonicity of the solution isadjusted to a value of about ⅓ to 3, more typically ½ to 2, and mostoften ¾ to 1.7.

The biologically active agent may be dispersed in a base or vehicle,which may comprise a hydrophilic compound having a capacity to dispersethe active agent and any desired additives. The base may be selectedfrom a wide range of suitable carriers, including but not limited to,copolymers of polycarboxylic acids or salts thereof, carboxylicanhydrides (e.g. maleic anhydride) with other monomers (e.g. methyl(meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such aspolyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulosederivatives such as hydroxymethylcellulose, hydroxypropylcellulose,etc., and natural polymers such as chitosan, collagen, sodium alginate,gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, abiodegradable polymer is selected as a base or carrier, for example,polylactic acid, poly(lactic acid-glycolic acid) copolymer,polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid)copolymer and mixtures thereof. Alternatively or additionally, syntheticfatty acid esters such as polyglycerin fatty acid esters, sucrose fattyacid esters, etc. can be employed as carriers. Hydrophilic polymers andother carriers can be used alone or in combination, and enhancedstructural integrity can be imparted to the carrier by partialcrystallization, ionic bonding, crosslinking and the like. The carriercan be provided in a variety of forms, including, fluid or viscoussolutions, gels, pastes, powders, microspheres and films for directapplication to the nasal mucosa. The use of a selected carrier in thiscontext may result in promotion of absorption of the biologically activeagent.

The biologically active agent can be combined with the base or carrieraccording to a variety of methods, and release of the active agent maybe by diffusion, disintegration of the carrier, or associatedformulation of water channels. In some circumstances, the active agentis dispersed in microcapsules (microspheres) or nanocapsules(nanospheres) prepared from a suitable polymer, e.g., isobutyl2-cyanoacrylate (see, e.g., Michael, et al., J. Pharmacy Pharmacol.43:1-5, 1991), and dispersed in a biocompatible dispersing mediumapplied to the nasal mucosa, which yields sustained delivery andbiological activity over a protracted time.

To further enhance mucosal delivery of pharmaceutical agents within theinvention, formulations comprising the active agent may also contain ahydrophilic low molecular weight compound as a base or excipient. Suchhydrophilic low molecular weight compounds provide a passage mediumthrough which a water-soluble active agent, such as a physiologicallyactive peptide or protein, may diffuse through the base to the bodysurface where the active agent is absorbed. The hydrophilic lowmolecular weight compound optionally absorbs moisture from the mucosa orthe administration atmosphere and dissolves the water-soluble activepeptide. The molecular weight of the hydrophilic low molecular weightcompound is generally not more than 10000 and preferably not more than3000. Exemplary hydrophilic low molecular weight compound include polyolcompounds, such as oligo-, di- and monosaccarides such as sucrose,mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose,D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerin andpolyethylene glycol. Other examples of hydrophilic low molecular weightcompounds useful as carriers within the invention includeN-methylpyrrolidone, and alcohols (e.g., oligovinyl alcohol, ethanol,ethylene glycol, propylene glycol, etc.). These hydrophilic lowmolecular weight compounds can be used alone or in combination with oneanother or with other active or inactive components of the intranasalformulation.

The compositions of the invention may alternatively contain aspharmaceutically acceptable carriers substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc. For solid compositions, conventional nontoxicpharmaceutically acceptable carriers can be used which include, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

Therapeutic compositions for administering the biologically active agentcan also be formulated as a solution, microemulsion, or other orderedstructure suitable for high concentration of active ingredients. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. Proper fluidity for solutions can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of adesired particle size in the case of dispersible formulations, and bythe use of surfactants. In many cases, it will be desirable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe biologically active agent can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin.

In certain embodiments of the invention, the biologically active agentis administered in a time release formulation, for example in acomposition which includes a slow release polymer. The active agent canbe prepared with carriers that will protect against rapid release, forexample a controlled release vehicle such as a polymer,microencapsulated delivery system or bioadhesive gel. Prolonged deliveryof the active agent, in various compositions of the invention can bebrought about by including in the composition agents that delayabsorption, for example, aluminum monosterate hydrogels and gelatin.When controlled release formulations of the biologically active agent isdesired, controlled release binders suitable for use in accordance withthe invention include any biocompatible controlled-release materialwhich is inert to the active agent and which is capable of incorporatingthe biologically active agent. Numerous such materials are known in theart. Useful controlled-release binders are materials that aremetabolized slowly under physiological conditions following theirintranasal delivery (e.g., at the nasal mucosal surface, or in thepresence of bodily fluids following transmucosal delivery). Appropriatebinders include but are not limited to biocompatible polymers andcopolymers previously used in the art in sustained release formulations.Such biocompatible compounds are non-toxic and inert to surroundingtissues, and do not trigger significant adverse side effects such asnasal irritation, immune response, inflammation, or the like. They aremetabolized into metabolic products that are also biocompatible andeasily eliminated from the body.

Exemplary polymeric materials for use in this context include, but arenot limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolysable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids (PGA) and polylactic acids (PLA),poly(DL-lactic acid-co-glycolic acid) (DL PLGA), poly(D-lacticacid-coglycolic acid) (D PLGA) and poly(L-lactic acid-co-glycolic acid)(L PLGA). Other useful biodegradable or bioerodable polymers include butare not limited to such polymers as poly(epsilon-caprolactone),poly(epsilon-aprolactone-CO-lactic acid), poly(ε-aprolactone-CO-glycolicacid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate),hydrogels such as poly(hydroxyethyl methacrylate), polyamides,poly(amino acids) (i.e., L-leucine, glutamic acid, L-aspartic acid andthe like), poly (ester urea), poly (2-hydroxyethyl DL-aspartamide),polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides,polysaccharides and copolymers thereof. Many methods for preparing suchformulations are generally known to those skilled in the art. J. R.Robinson, ed., Sustained and Controlled Release Drug Delivery Systems,Marcel Dekker, Inc., New York, 1978, incorporated herein by reference.Other useful formulations include controlled-release compositions suchas are known in the art for the administration of leuprolide (tradename: Lupron®), e.g., microcapsules, U.S. Pat. Nos. 4,652,441 and4,917,893, each incorporated herein by reference, lactic acid-glycolicacid copolymers useful in making microcapsules and other formulations,U.S. Pat. Nos. 4,677,191 and 4,728,721, each incorporated herein byreference, and sustained-release compositions for water-solublepeptides. U.S. Pat. No. 4,675,189, incorporated herein by reference.

The mucosal formulations of the invention typically must be sterile andstable under all conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the active compound in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The prevention of the action ofmicroorganisms can be accomplished by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like.

In more detailed aspects of the invention, the biologically active agentis stabilized to extend its effective half-life following delivery tothe subject, particularly for extending metabolic persistence in anactive state within the physiological environment (e.g., at the nasalmucosal surface, in the bloodstream, or within a connective tissuecompartment or fluid-filled body cavity). For this purpose, thebiologically active agent may be modified by chemical means, e.g.,chemical conjugation, N-terminal capping, PEGylation, or recombinantmeans, e.g., site-directed mutagenesis or construction of fusionproteins, or formulated with various stabilizing agents or carriers.Thus stabilized, the active agent administered as above retainsbiological activity for an extended period (e.g., 2-3, up to 5-10 foldgreater stability) under physiological conditions compared to itsnon-stabilized form.

In accordance with the various treatment methods of the invention, thebiologically active agent is delivered to a mammalian subject in amanner consistent with conventional methodologies associated withmanagement of the disorder for which treatment or prevention is sought.In accordance with the disclosure herein, a prophylactically ortherapeutically effective amount of the biologically active agent isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent, inhibit, and/or ameliorate a selecteddisease or condition or one or more symptom(s) thereof.

The term “subject” as used herein means any mammalian patient to whichthe compositions of the invention may be administered. Typical subjectsintended for treatment with the compositions and methods of the presentinvention include humans, as well as non-human primates and otheranimals. To identify subject patients for prophylaxis or treatmentaccording to the methods of the invention, accepted screening methodsare employed to determine risk factors associated with a targeted orsuspected disease of condition as discussed above, or to determine thestatus of an existing disease or condition in a subject. These screeningmethods include, for example, conventional work-ups to determinefamilial, sexual, drug-use and other such risk factors that may beassociated with the targeted or suspected disease or condition, as wellas diagnostic methods such as various ELISA immunoassay methods, whichare available and well known in the art to detect and/or characterizedisease-associated markers. These and other routine methods allow theclinician to select patients in need of therapy using the mucosalmethods and formulations of the invention. In accordance with thesemethods and principles, biologically active agents may be mucosallyadministered according to the teachings herein as an independentprophylaxis or treatment program, or as a follow-up, adjunct orcoordinate treatment regimen to other treatments, including surgery,vaccination, immunotherapy, hormone treatment, cell, tissue, or organtransplants, and the like.

Mucosal administration according to the invention allows effectiveself-administration of treatment by patients, provided that sufficientsafeguards are in place to control and monitor dosing and side effects.Mucosal administration also overcomes certain drawbacks of otheradministration forms, such as injections, that are painful and exposethe patient to possible infections and may present drug bioavailabilityproblems. For nasal and pulmonary delivery, systems for controlledaerosol dispensing of therapeutic liquids as a spray are well known. Inone embodiment, metered doses of active agent are delivered by means ofa specially constructed mechanical pump valve (U.S. Pat. No. 4,511,069,incorporated herein by reference). This hand-held delivery device isuniquely nonvented so that sterility of the solution in the aerosolcontainer is maintained indefinitely.

Dosage

For prophylactic and treatment purposes, the biologically activeagent(s) disclosed herein may be administered to the subject in a singlebolus delivery, via continuous delivery (e.g., continuous transdermal,mucosal, or intravenous delivery) over an extended time period, or in arepeated administration protocol (e.g., by an hourly, daily or weekly,repeated administration protocol). In this context, a therapeuticallyeffective dosage of the biologically active agent(s) may includerepeated doses within a prolonged prophylaxis or treatment regimen, thatwill yield clinically significant results to alleviate one or moresymptoms or detectable conditions associated with a targeted disease orcondition as set forth above. Determination of effective dosages in thiscontext is typically based on animal model studies followed up by humanclinical trials and is guided by determining effective dosages andadministration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject.Suitable models in this regard include, for example, murine, rat,porcine, feline, non-human primate, and other accepted animal modelsubjects known in the art. Alternatively, effective dosages can bedetermined using in vitro models (e.g., immunologic and histopathologicassays). Using such models, only ordinary calculations and adjustmentsare typically required to determine an appropriate concentration anddose to administer a therapeutically effective amount of thebiologically active agent(s) (e.g., amounts that are intranasallyeffective, transdermally effective, intravenously effective, orintramuscularly effective to elicit a desired response). In alternativeembodiments, an “effective amount” or “effective dose” of thebiologically active agent(s) may simply inhibit or enhance one or moreselected biological activity(ies) correlated with a disease orcondition, as set forth above, for either therapeutic or diagnosticpurposes.

The actual dosage of biologically active agents will of course varyaccording to factors such as the disease indication and particularstatus of the subject (e.g., the subject's age, size, fitness, extent ofsymptoms, susceptibility factors, etc.), time and route ofadministration, other drugs or treatments being administeredconcurrently, as well as the specific pharmacology of the biologicallyactive agent(s) for eliciting the desired activity or biologicalresponse in the subject. Dosage regimens may be adjusted to provide anoptimum prophylactic or therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental sideeffects of the biologically active agent is outweighed in clinical termsby therapeutically beneficial effects. A non-limiting range for atherapeutically effective amount of a biologically active agent withinthe methods and formulations of the invention is 0.01 μg/kg-10 mg/kg,more typically between about 0.05 and 5 mg/kg, and in certainembodiments between about 0.2 and 2 mg/kg. Alternatively, a non-limitingrange for a therapeutically effective amount of a biologically activeagent within the methods and formulations of the invention is betweenabout 0.001 pmol to about 100 pmol per kg body weight, between about0.01 pmol to about 10 pmol per kg body weight, between about 0.1 pmol toabout 5 pmol per kg body weight, or between about 0.5 pmol to about 1.0pmol per kg body weight. Dosages within this range can be achieved bysingle or multiple administrations, including, e.g., multipleadministrations per day, daily or weekly administrations. Peradministration, it is desirable to administer at least one microgram ofthe biologically active agent (e.g., one or more peptide YY proteins,analogs and mimetics, and other biologically active agents), moretypically between about 10 μg and 5.0 mg, and in certain embodimentsbetween about 100 μg and 1.0 or 2.0 mg to an average human subject. Itis to be further noted that for each particular subject, specific dosageregimens should be evaluated and adjusted over time according to theindividual need and professional judgment of the person administering orsupervising the administration of the permeabilizing peptide(s) andother biologically active agent(s).

Dosage of biologically active agents may be varied by the attendingclinician to maintain a desired concentration at the target site. Forexample, a selected local concentration of the biologically active agentin the bloodstream or CNS may be about 1-50 nanomoles per liter,sometimes between about 1.0 nanomole per liter and 10, 15 or 25nanomoles per liter, depending on the subject's status and projected ormeasured response. In an alternative example, a selected localconcentration of the biologically active agent in the bloodstream or CNSmay be between about 0.1 pmol/L to about 1000 pmol/L of blood plasma orCSF, between about 1.0 pmol/L to about 100 pmol/L of blood plasma orCSF, between about 1.0 pmol/L to about 10 pmol/L of blood plasma or CSF,or between about 5.0 pmol/L to about 10 pmol/L of blood plasma or CSF.Higher or lower concentrations may be selected based on the mode ofdelivery, e.g., trans-epidermal, rectal, oral, or intranasal deliveryversus intravenous or subcutaneous delivery. Dosage should also beadjusted based on the release rate of the administered formulation,e.g., of a nasal spray versus powder, sustained release oral versusinjected particulate or transdermal delivery formulations, etc. Toachieve the same serum concentration level, for example, slow-releaseparticles with a release rate of 5 nanomolar (under standard conditions)would be administered at about twice the dosage of particles with arelease rate of 10 nanomolar.

Additional guidance as to particular dosages for selected biologicallyactive agents for use within the invention may be found widelydisseminated in the literature. This is true for many of the therapeuticpeptide and protein agents disclosed herein. For example, guidance foradministration of human growth hormone (hGH) in the treatment ofindividuals intoxicated with poisonous substances may be found in U.S.Pat. Nos. 5,140,008 and 4,816,439; guidance for administration of hGH inthe treatment of topical ulcers may be found in U.S. Pat. No. 5,006,509.

Kits

The instant invention also includes kits, packages and multicontainerunits containing the above described pharmaceutical compositions, activeingredients, and/or means for administering the same for use in theprevention and treatment of diseases and other conditions in mammaliansubjects. Briefly, these kits include a container or formulation thatcontains one or more peptide YY proteins, analogs or mimetics, and/orother biologically active agents in combination with mucosal deliveryenhancing agents disclosed herein formulated in a pharmaceuticalpreparation for mucosal delivery. The biologically active agent(s)is/are optionally contained in a bulk dispensing container or unit ormulti-unit dosage form. Optional dispensing means may be provided, forexample a pulmonary or intranasal spray applicator. Packaging materialsoptionally include a label or instruction indicating that thepharmaceutical agent packaged therewith can be used mucosally, e.g.,intranasally, for treating or preventing a specific disease orcondition. In more detailed embodiments of the invention, kits includeone or more mucosal delivery-enhancing agents selected from: (a)aggregation inhibitory agents; (b) charge modifying agents; (c) pHcontrol agents; (d) degradative enzyme inhibitors; (e) mucolytic ormucus clearing agents; (f) ciliostatic agents; (g) membranepenetration-enhancing agents (e.g., (i) a surfactant, (ii) a bile salt,(iii) a phospholipid or fatty acid additive, mixed micelle, liposome, orcarrier, (iv) an alcohol, (v) an enamine, (vi) an NO donor compound,(vii) a long-chain amphipathic molecule, (viii) a small hydrophobicpenetration enhancer; (ix) sodium or a salicylic acid derivative; (x) aglycerol ester of acetoacetic acid, (xi) a cyclodextrin orbeta-cyclodextrin derivative, (xii) a medium-chain fatty acid, (xiii) achelating agent, (xiv) an amino acid or salt thereof, (xv) anN-acetylamino acid or salt thereof, (xvi) an enzyme degradative to aselected membrane component, (xvii) an inhibitor of fatty acidsynthesis, (xviii) an inhibitor of cholesterol synthesis; or (xix) anycombination of the membrane penetration enhancing agents of(i)-(xviii)); (h) secondary modulatory agents of epithelial junctionphysiology, such as nitric oxide (NO) stimulators, chitosan, andchitosan derivatives; (i) vasodilator agents; (j) selectivetransport-enhancing agents; and (k) stabilizing delivery vehicles,carriers, supports or complex-forming species with which thebiologically active agent is/are effectively combined, associated,contained, encapsulated or bound to stabilize the active agent forenhanced mucosal delivery.

The following examples are provided by way of illustration, notlimitation.

EXAMPLE 1

An exemplary formulation for enhanced nasal mucosal delivery of peptideYY following the teachings of the instant specification was prepared andevaluated as follows:

TABLE 1 Peptide YY Formulation Composition Peptide YY₃₋₃₆ FormulationsPer 100 ml Sample Mucosal Delivery Enhancing Agent A 60 μgPhosphate-buffered saline (0.8%) pH 7.4 (Control 1) B 60 μgPhosphate-buffered saline (0.8%) pH 5.0 (Control 2) C 60 μg L-Arginine(10% w/v) D 60 μg Poly-L-Arginine (0.5% w/v) E 60 μg Gamma-Cyclodextrin(1% w/v) F 60 μg α-Cyclodextrin (5% w/v) G 60 μg Methyl-β-Cyclodextrin(3% w/v) H 60 μg n-Capric Acid Sodium (0.075% w/v) I 60 μg Chitosan(0.5% w/v) J 60 μg L-α-phosphatidilcholine didecanyl (3.5% w/v) K 60 μgS-Nitroso-N-Acetyl-Penicillamine (0.5% w/v) L 60 μgPalmotoyl-DL-Carnitine (0.02% w/v) M 60 μg Pluronic-127 (0.3% w/v) N 60μg Sodium Nitroprusside (0.3% w/v) O 60 μg Sodium Glycocholate (1% w/v)P 60 μg F1: Gelatin, DDPC, MBCD, EDTA F 1 L-α-phosphatidilcholinedidecanyl (0.5% w/v) Methyl β Cyclodextrin (3% w/v) EDTA (0.1% w/v, Inf.Conc. 0.5M) Gelatin (0.5% w/v)

EXAMPLE 2 Nasal Mucosal Delivery Permeation Kinetics and Cytotoxicity

1. Organotypic Model

The following methods are generally useful for evaluating nasal mucosaldelivery parameters, kinetics and side effects for peptide YY within theformulations and method of the invention, as well as for determining theefficacy and characteristics of the various intranasaldelivery-enhancing agents disclosed herein for combinatorial formulationor coordinate administration with peptide YY.

Permeation kinetics and cytotoxicity are also useful for determining theefficacy and characteristics of the various mucosal delivery-enhancingagents disclosed herein for combinatorial formulation or coordinateadministration with mucosal delivery-enhancing agents. In one exemplaryprotocol, permeation kinetics and lack of unacceptable cytotoxicity aredemonstrated for an intranasal delivery-enhancing agents as disclosedabove in combination with a biologically active therapeutic agent,exemplified by peptide YY.

The EpiAirway system was developed by MatTek Corp (Ashland, Mass.) as amodel of the pseudostratified epithelium lining the respiratory tract.The epithelial cells are grown on porous membrane-bottomed cell cultureinserts at an air-liquid interface, which results in differentiation ofthe cells to a highly polarized morphology. The apical surface isciliated with a microvillous ultrastructure and the epithelium producesmucus (the presence of mucin has been confirmed by immunoblotting). Theinserts have a diameter of 0.875 cm, providing a surface area of 0.6cm². The cells are plated onto the inserts at the factory approximatelythree weeks before shipping. One “kit” consists of 24 units.

-   -   A. On arrival, the units are placed onto sterile supports in        6-well microplates. Each well receives 5 mL of proprietary        culture medium. This DMEM-based medium is serum free but is        supplemented with epidermal growth factor and other factors. The        medium is always tested for endogenous levels of any cytokine or        growth factor which is being considered for intranasal delivery,        but has been free of all cytokines and factors studied to date        except insulin. The 5 mL volume is just sufficient to provide        contact to the bottoms of the units on their stands, but the        apical surface of the epithelium is allowed to remain in direct        contact with air. Sterile tweezers are used in this step and in        all subsequent steps involving transfer of units to        liquid-containing wells to ensure that no air is trapped between        the bottoms of the units and the medium.    -   B. The units in their plates are maintained at 37° C. in an        incubator in an atmosphere of 5% CO₂ in air for 24 hours. At the        end of this time the medium is replaced with fresh medium and        the units are returned to the incubator for another 24 hours.

2. Experimental Protocol—Permeation Kinetics

-   -   A. A “kit” of 24 EpiAirway units can routinely be employed for        evaluating five different formulations, each of which is applied        to quadruplicate wells. Each well is employed for determination        of permeation kinetics (4 time points), transepithelial        resistance, mitochondrial reductase activity as measured by MTT        reduction, and cytolysis as measured by release of LDH. An        additional set of wells is employed as controls, which are sham        treated during determination of permeation kinetics, but are        otherwise handled identically to the test sample-containing        units for determinations of transepithelial resistance and        viability. The determinations on the controls are routinely also        made on quadruplicate units, but occasionally we have employed        triplicate units for the controls and have dedicated the        remaining four units in the kit to measurements of        transepithelial resistance and viability on untreated units or        we have frozen and thawed the units for determinations of total        LDH levels to serve as a reference for 100% cytolysis.    -   B. In all experiments, the nasal mucosal delivery formulation to        be studied is applied to the apical surface of each unit in a        volume of 100 μL, which is sufficient to cover the entire apical        surface. An appropriate volume of the test formulation at the        concentration applied to the apical surface (no more than 100 μL        is generally needed) is set aside for subsequent determination        of concentration of the active material by ELISA or other        designated assay.    -   C. The units are placed in 6 well plates without stands for the        experiment: each well contains 0.9 mL of medium which is        sufficient to contact the porous membrane bottom of the unit but        does not generate any significant upward hydrostatic pressure on        the unit.    -   D. In order to minimize potential sources of error and avoid any        formation of concentration gradients, the units are transferred        from one 0.9 mL-containing well to another at each time point in        the study. These transfers are made at the following time        points, based on a zero time at which the 100 μL volume of test        material was applied to the apical surface: 15 minutes, 30        minutes, 60 minutes, and 120 minutes.    -   E. In between time points the units in their plates are kept in        the 37° C. incubator. Plates containing 0.9 mL medium per well        are also maintained in the incubator so that minimal change in        temperature occurs during the brief periods when the plates are        removed and the units are transferred from one well to another        using sterile forceps.    -   F. At the completion of each time point, the medium is removed        from the well from which each unit was transferred, and        aliquotted into two tubes (one tube receives 700 μL and the        other 200 μL) for determination of the concentration of        permeated test material and, in the event that the test material        is cytotoxic, for release of the cytosolic enzyme, lactic        dehydrogenase, from the epithelium. These samples are kept in        the refrigerator if the assays are to be conducted within 24        hours, or the samples are subaliquotted and kept frozen at        −80° C. until thawed once for assays. Repeated freeze-thaw        cycles are to be avoided.    -   G. In order to minimize errors, all tubes, plates, and wells are        prelabeled before initiating an experiment.    -   H. At the end of the 120 minute time point, the units are        transferred from the last of the 0.9 mL containing wells to        24-well microplates, containing 0.3 mL medium per well. This        volume is again sufficient to contact the bottoms of the units,        but not to exert upward hydrostatic pressure on the units. The        units are returned to the incubator prior to measurement of        transepithelial resistance.

3. Experimental Protocol—Transepithelial Resistance

-   -   A. Respiratory airway epithelial cells form tight junctions in        vivo as well as in vitro, restricting the flow of solutes across        the tissue. These junctions confer a transepithelial resistance        of several hundred ohms×cm² in excised airway tissues; in the        MatTek EpiAirway units, the transepithelial resistance (TER) is        claimed by the manufacturer to be routinely around 1000        ohms×cm². We have found that the TER of control EpiAirway units        which have been sham-exposed during the sequence of steps in the        permeation study is somewhat lower (700-800 ohms×cm²), but,        since permeation of small molecules is proportional to the        inverse of the TER, this value is still sufficiently high to        provide a major barrier to permeation. The porous        membrane-bottomed units without cells, conversely, provide only        minimal transmembrane resistance (5-20 ohms×cm²).    -   B. Accurate determinations of TER require that the electrodes of        the ohmmeter be positioned over a significant surface area above        and below the membrane, and that the distance of the electrodes        from the membrane be reproducibly controlled. The method for TER        determination recommended by MatTek and employed for all        experiments here employs an “EVOM”™ epithelial voltohmmeter and        an “ENDOHM”™ tissue resistance measurement chamber from World        Precision Instruments, Inc., Sarasota, Fla.    -   C. The chamber is initially filled with Dulbecco's phosphate        buffered saline (PBS) for at least 20 minutes prior to TER        determinations in order to equilibrate the electrodes.    -   D. Determinations of TER are made with 1.5 mL of PBS in the        chamber and 350 μL of PBS in the membrane-bottomed unit being        measured. The top electrode is adjusted to a position just above        the membrane of a unit containing no cells (but containing 350        μL of PBS) and then fixed to ensure reproducible positioning.        The resistance of a cell-free unit is typically 5-20 ohms×cm²        (“background resistance”).    -   E. Once the chamber is prepared and the background resistance is        recorded, units in a 24-well plate which had just been employed        in permeation determinations are removed from the incubator and        individually placed in the chamber for TER determinations.    -   F. Each unit is first transferred to a petri dish containing PBS        to ensure that the membrane bottom is moistened. An aliquot of        350 μL PBS is added to the unit and then carefully aspirated        into a labeled tube to rinse the apical surface. A second wash        of 350 μL PBS is then applied to the unit and aspirated into the        same collection tube.    -   G. The unit is gently blotted free of excess PBS on its exterior        surface only before being placed into the chamber (containing a        fresh 1.5 mL aliquot of PBS). An aliquot of 350 μL PBS is added        to the unit before the top electrode is placed on the chamber        and the TER is read on the EVOM meter.    -   H. After the TER of the unit is read in the ENDOHM chamber, the        unit is removed, the PBS is aspirated and saved, and the unit is        returned with an air interface on the apical surface to a        24-well plate containing 0.3 mL medium per well.    -   I. The units are read in the following sequence: all        sham-treated controls, followed by all formulation-treated        samples, followed by a second TER reading of each of the        sham-treated controls. After all the TER determinations are        complete, the units in the 24-well microplate are returned to        the incubator for determination of viability by MTT reduction.

4. Experimental Protocol—Viability by MTT Reduction

MTT is a cell-permeable tetrazolium salt which is reduced bymitochondrial dehydrogenase activity to an insoluble colored formazan byviable cells with intact mitochondrial function or by nonmitochondrialNAD(P)H dehydrogenase activity from cells capable of generating arespiratory burst. Formation of formazan is a good indicator ofviability of epithelial cells since these cells do not generate asignificant respiratory burst. We have employed a MTT reagent kitprepared by MatTek Corp for their units in order to assess viability.

-   -   A. The MTT reagent is supplied as a concentrate and is diluted        into a proprietary DMEM-based diluent on the day viability is to        be assayed (typically the afternoon of the day in which        permeation kinetics and TER were determined in the morning).        Insoluble reagent is removed by a brief centrifugation before        use. The final MTT concentration is 1 mg/mL.    -   B. The final MTT solution is added to wells of a 24-well        microplate at a volume of 300 μL per well. As has been noted        above, this volume is sufficient to contact the membranes of the        EpiAirway units but imposes no significant positive hydrostatic        pressure on the cells.    -   C. The units are removed from the 24-well plate in which they        were placed after TER measurements, and after removing any        excess liquid from the exterior surface of the units, they are        transferred to the plate containing MTT reagent. The units in        the plate are then placed in an incubator at 37° C. in an        atmosphere of 5% CO₂ in air for 3 hours.    -   D. At the end of the 3-hour incubation, the units containing        viable cells will have turned visibly purple. The insoluble        formazan must be extracted from the cells in their units to        quantitate the extent of MTT reduction. Extraction of the        formazan is accomplished by transferring the units to a 24-well        microplate containing 2 mL extractant solution per well, after        removing excess liquid from the exterior surface of the units as        before. This volume is sufficient to completely cover both the        membrane and the apical surface of the units. Extraction is        allowed to proceed overnight at room temperature in a        light-tight chamber. MTT extractants traditionally contain high        concentrations of detergent, and destroy the cells.    -   E. At the end of the extraction, the fluid from within each unit        and the fluid in its surrounding well are combined and        transferred to a tube for subsequent aliquotting into a 96-well        microplate (200 μL aliquots are optimal) and determination of        absorbance at 570 nm on a VMax multiwell microplate        spectrophotometer. To ensure that turbidity from debris coming        from the extracted units does not contribute to the absorbance,        the absorbance at 650 nm is also determined for each well in the        VMax and is automatically subtracted from the absorbance at 570        nm. The “blank” for the determination of formazan absorbance is        a 200 μL aliquot of extractant to which no unit had been        exposed. This absorbance value is assumed to constitute zero        viability.    -   F. Two units from each kit of 24 EpiAirway units are left        untreated during determination of permeation kinetics and TER.        These units are employed as the positive control for 100% cell        viability. In all the studies we have conducted, there has been        no statistically significant difference in the viability of the        cells in these untreated units vs cells in control units which        had been sham treated for permeation kinetics and on which TER        determinations had been performed. The absorbance of all units        treated with test formulations is assumed to be linearly        proportional to the percent viability of the cells in the units        at the time of the incubation with MTT. It should be noted that        this assay is carried out typically no sooner than four hours        after introduction of the test material to the apical surface,        and subsequent to rinsing of the apical surface of the units        during TER determination.

5. Determination of Viability by LDH Release

While measurement of mitochondrial reductase activity by MTT reductionis a sensitive probe of cell viability, the assay necessarily destroysthe cells and therefore can be carried out only at the end of eachstudy. When cells undergo necrotic lysis, their cytotosolic contents arespilled into the surrounding medium, and cytosolic enzymes such aslactic dehydrogenase (LDH) can be detected in this medium. An assay forLDH in the medium can be performed on samples of medium removed at eachtime point of the two-hour determination of permeation kinetics. Thus,cytotoxic effects of formulations which do not develop until significanttime has passed can be detected as well as effects of formulations whichinduce cytolysis with the first few minutes of exposure to airwayepithelium.

-   -   A. The recommended LDH assay for evaluating cytolysis of the        EpiAirway units is based on conversion of lactate to pyruvate        with generation of NADH from NAD. The NADH is then reoxidized        along with simultaneous reduction of the tetrazolium salt INT,        catalyzed by a crude “diaphorase” preparation. The formazan        formed from reduction of INT is soluble, so that the entire        assay for LDH activity can be carried out in a homogenous        aqueous medium containing lactate, NAD, diaphorase, and INT.    -   B. The assay for LDH activity is carried out on 50 μL aliquots        from samples of “supernatant” medium surrounding an EpiAirway        unit and collected at each time point. These samples were either        stored for no longer than 24 h in the refrigerator or were        thawed after being frozen within a few hours after collection.        Each EpiAirway unit generates samples of supernatant medium        collected at 15 min, 30 min, 1 h, and 2 h after application of        the test material. The aliquots are all transferred to a 96 well        microplate.    -   C. A 50 μL aliquot of medium which had not been exposed to a        unit serves as a “blank” or negative control of 0% cytotoxicity.        We have found that the apparent level of “endogenous” LDH        present after reaction of the assay reagent mixture with the        unexposed medium is the same within experimental error as the        apparent level of LDH released by all the sham-treated control        units over the entire time course of 2 hours required to conduct        a permeation kinetics study. Thus, within experimental error,        these sham-treated units show no cytolysis of the epithelial        cells over the time course of the permeation kinetics        measurements.    -   D. To prepare a sample of supernatant medium reflecting the        level of LDH released after 100% of the cells in a unit have        lysed, a unit which had not been subjected to any prior        manipulations is added to a well of a 6-well microplate        containing 0.9 mL of medium as in the protocol for determination        of permeation kinetics, the plate containing the unit is frozen        at −80° C., and the contents of the well are then allowed to        thaw. This freeze-thaw cycle effectively lyses the cells and        releases their cytosolic contents, including LDH, into the        supernatant medium. A 50 μL aliquot of the medium from the        frozen and thawed cells is added to the 96-well plate as a        positive control reflecting 100% cytotoxicity.    -   E. To each well containing an aliquot of supernatant medium, a        50 μL aliquot of the LDH assay reagent is added. The plate is        then incubated for 30 minutes in the dark.    -   F. The reactions are terminated by addition of a “stop” solution        of 1 M acetic acid, and within one hour of addition of the stop        solution, the absorbance of the plate is determined at 490 nm.    -   G. Computation of percent cytolysis is based on the assumption        of a linear relationship between absorbance and cytolysis, with        the absorbance obtained from the medium alone serving as a        reference for 0% cytolysis and the absorbance obtained from the        medium surrounding a frozen and thawed unit serving as a        reference for 100% cytolysis.

6 ELISA Determinations

The procedures for determining the concentrations of biologically activeagents as test materials for evaluating enhanced permeation of activeagents in conjunction with coordinate administration of mucosaldelivery-enhancing agents or combinatorial formulation of the inventionare generally as described above and in accordance with known methodsand specific manufacturer instructions of ELISA kits employed for eachparticular assay. Permeation kinetics of the biologically active agentis generally determined by taking measurements at multiple time points(for example 15 min., 30 min., 60 min. and 120 min) after thebiologically active agent is contacted with the apical epithelial cellsurface (which may be simultaneous with, or subsequent to, exposure ofthe apical cell surface to the mucosal delivery-enhancing agent(s)).

The procedures for determining the concentrations of peptide YYneuropeptide Y, and pancreatic peptide in blood serum, central nervoussystem (CNS) tissues or fluids, cerebral spinal fluid (CSF), or othertissues or fluids of a mammalian subject may be determined byimmunologic assay for peptide YY neuropeptide Y, and pancreatic peptide.The procedures for determining the concentrations of peptide YYneuropeptide Y, and pancreatic peptide as test materials for evaluatingenhanced permeation of active agents in conjunction with coordinateadministration of mucosal delivery-enhancing agents or combinatorialformulation of the invention are generally as described above and inaccordance with known methods and specific manufacturer instructions forradioimmunoassay (RIA), enzyme immunoassay (EIA), and antibody reagentsfor immunohistochemistry or immunofluorescence for peptide YYneuropeptide Y, and pancreatic peptide. Bachem AG (King of Prussia,Pa.).

EpiAirway™ tissue membranes are cultured in phenol red andhydrocortisone free medium (MatTek Corp., Ashland, Mass.). The tissuemembranes are cultured at 37° C. for 48 hours to allow the tissues toequilibrate. Each tissue membrane is placed in an individual well of a6-well plate containing 0.9 mL of serum free medium. 100 μL of theformulation (test sample or control) is applied to the apical surface ofthe membrane. Triplicate or quadruplicate samples of each test sample(mucosal delivery-enhancing agent in combination with a biologicallyactive agent, peptide YY) and control (biologically active agent,peptide YY, alone) are evaluated in each assay. At each time point (15,30, 60 and 120 minutes) the tissue membranes are moved to new wellscontaining fresh medium. The underlying 0.9 mL medium samples isharvested at each time point and stored at 4° C. for use in ELISA andlactate dehydrogenase (LDH) assays.

The ELISA kits are typically two-step sandwich ELISAs: theimmunoreactive form of the agent being studied is first “captured” by anantibody immobilized on a 96-well microplate and after washing unboundmaterial out of the wells, a “detection” antibody is allowed to reactwith the bound immunoreactive agent. This detection antibody istypically conjugated to an enzyme (most often horseradish peroxidase)and the amount of enzyme bound to the plate in immune complexes is thenmeasured by assaying its activity with a chromogenic reagent. Inaddition to samples of supernatant medium collected at each of the timepoints in the permeation kinetics studies, appropriately diluted samplesof the formulation (i.e., containing the subject biologically activetest agent) that was applied to the apical surface of the units at thestart of the kinetics study are also assayed in the ELISA plate, alongwith a set of manufacturer-provided standards. Each supernatant mediumsample is generally assayed in duplicate wells by ELISA (it will berecalled that quadruplicate units are employed for each formulation in apermeation kinetics determination, generating a total of sixteen samplesof supernatant medium collected over all four time points).

-   -   A. It is not uncommon for the apparent concentrations of active        test agent in samples of supernatant medium or in diluted        samples of material applied to the apical surface of the units        to lie outside the range of concentrations of the standards        after completion of an ELISA. No concentrations of material        present in experimental samples are determined by extrapolation        beyond the concentrations of the standards; rather, samples are        rediluted appropriately to generate concentrations of the test        material which can be more accurately determined by        interpolation between the standards in a repeat ELISA.    -   B. The ELISA for a biologically active test agent, for example,        peptide YY, is unique in its design and recommended protocol.        Unlike most kits, the ELISA employs two monoclonal antibodies,        one for capture and another, directed towards a nonoverlapping        determinant for the biologically active test agent, e.g.,        peptide YY, as the detection antibody (this antibody is        conjugated to horseradish peroxidase). As long as concentrations        of peptide YY that lie below the upper limit of the assay are        present in experimental samples, the assay protocol can be        employed as per the manufacturer's instructions, which allow for        incubation of the samples on the ELISA plate with both        antibodies present simultaneously. When the peptide YY levels in        a sample are significantly higher than this upper limit, the        levels of immunoreactive peptide YY may exceed the amounts of        the antibodies in the incubation mixture, and some peptide YY        which has no detection antibody bound will be captured on the        plate, while some peptide YY which has detection antibody bound        may not be captured. This leads to serious underestimation of        the peptide YY levels in the sample (it will appear that the        peptide YY levels in such a sample lie significantly below the        upper limit of the assay). To eliminate this possibility, the        assay protocol has been modified:    -   B.1. The diluted samples are first incubated on the ELISA plate        containing the immobilized capture antibody for one hour in the        absence of any detection antibody. After the one hour        incubation, the wells are washed free of unbound material.    -   B.2. The detection antibody is incubated with the plate for one        hour to permit formation of immune complexes with all captured        antigen. The concentration of detection antibody is sufficient        to react with the maximum level of peptide YY which has been        bound by the capture antibody. The plate is then washed again to        remove any unbound detection antibody.    -   B.3. The peroxidase substrate is added to the plate and        incubated for fifteen minutes to allow color development to take        place.    -   B.4. The “stop” solution is added to the plate, and the        absorbance is read at 450 nm as well as 490 nm in the VMax        microplate spectrophotometer. The absorbance of the colored        product at 490 nm is much lower than that at 450 nm, but the        absorbance at each wavelength is still proportional to        concentration of product. The two readings ensure that the        absorbance is linearly related to the amount of bound peptide YY        over the working range of the VMax instrument (we routinely        restrict the range from 0 to 2.5 OD, although the instrument is        reported to be accurate over a range from 0 to 3.0 OD). The        amount of peptide YY in the samples is determined by        interpolation between the OD values obtained for the different        standards included in the ELISA. Samples with OD readings        outside the range obtained for the standards are rediluted and        run in a repeat ELISA.

RESULTS

Measurement of Trans Epithelial Resistance by TER Assay:

After the final assay time points, membranes were placed in individualwells of a 24 well culture plate in 0.3 mL of clean medium and the transepithelial electrical resistance (TER) was measured using the EVOMEpithelial Voltohmmeter and an Endohm chamber (World PrecisionInstruments, Sarasota, Fla.). The top electrode was adjusted to be closeto, but not in contact with, the top surface of the membrane. Tissueswere removed, one at a time, from their respective wells and basalsurfaces were rinsed by dipping in clean PBS. Apical surfaces weregently rinsed twice with PBS. The tissue unit was placed in the Endohmchamber, 250 μL of PBS added to the insert, the top electrode replacedand the resistance measured and recorded. Following measurement, the PBSwas decanted and the tissue insert was returned to the culture plate.All TER values are reported as a function of the surface area of thetissue.

The final numbers were calculated as:TER of cell membrane=(Resistance (R) of Insert with membrane−R of blankInsert)×Area of membrane (0.6 cm²).

The effect of pharmaceutical formulations comprising peptide YY andintranasal delivery-enhancing agents on TER measurements across theEpiAirway™ Cell Membrane (mucosal epithelial cell layer) were tested. Adecrease in TER value relative to the control value(control=approximately 1000 ohms-cm²; normalized to 100.) indicates adecrease in cell membrane resistance and an increase in mucosalepithelial cell permeability.

Exemplary peptide YY formulation, Formulation P, showed the greatestdecrease in cell membrane resistance. (Table 2). The results indicatethat the exemplary formulation (e.g., Formulation P) reduces theresistance of the membrane to less than 1% of the control at theconcentrations tested. The values shown are the average of threereplicates of each formulation. Formulations A and B are controlsprepared by reconstituting peptide YY (Bachem AG, King of Prussia, Pa.)containing 60 μg peptide Y₃₋₃₆ in 100 ml of phosphate buffered saline(PBS) at pH 7.4 or 5.0. Peptide YY without mucosal delivery enhancersdid not decrease the resistance.

The results indicate that an exemplary formulation for enhancedintranasal delivery of peptide YY (e.g., Formulation P) decreases cellmembrane resistance and significantly increases mucosal epithelial cellspermeability. The exemplary formulations will enhance intranasaldelivery of peptide YY to the blood serum or to the central nervoussystem tissue or fluid. The results indicate that these exemplaryformulations when contacted with a mucosal epithelium yield significantincreases in mucosal epithelial cell permeability to peptide YY.

TABLE 2 Influence of Pharmaceutical Formulations Comprising Peptide YYand Intranasal Delivery-Enhancing Agents on Transepithelial Resistance(TER) of EpiAirway Cell Membrane Formulation Mucosal Delivery EnhancingAgent % TER A PBS pH 7.4 (Control 1) 100 B PBS pH 5.0 (Control 2) 100 CL-Arginine (10% w/v) 47.88 D Poly-L-Arginine (0.5% w/v) 3.96 EGamma-Cyclodextrin (1% w/v) 91.67 F Alpha-Cyclodextrin (5% w/v) 88.91 GMethyl-β-Cyclodextrin (3% w/v) 97.51 H n-Capric Acid Sodium (0.075% w/v)47.72 I Chitosan (0.5% w/v) 4.77 J L-α-phosphatidilcholine didecanyl(3.5% w/v) 0.49 K S-Nitroso-N-Acetyl-Penicillamine (0.5% w/v) 44.35 LPalmotoyl-DL-Carnitine (0.02% w/v) 1.76 M Pluronic-127 (0.3% w/v) 97.57N Sodium Nitroprusside (0.3% w/v) 92.41 O Sodium Glycocholate (1% w/v)14.25 P F1: Gelatin, DDPC, MBCD, EDTA 0.65Permeation Kinetics as Measured by ELISA Assay: The effect ofpharmaceutical formulations of the present invention comprising peptideYY and intranasal delivery-enhancing agents on the permeation of peptideYY across the EpiAirway™ Cell Membrane (mucosal epithelial cell layer)is measured as described above. The results are shown in Table 3.Permeation of peptide YY across the EpiAirway™ Cell Membrane is measuredby ELISA assay.

For the exemplary intranasal formulations (e.g., Formulation P) of thepresent invention, the greatest increase in peptide YY permeationoccurred in Formulation P as shown in Table 3. The procedure uses anELISA assay to determine the concentration of biologically activepeptide YY that has permeated the epithelial cells into the surroundingmedium over multiple time points. The results show increased permeationof peptide YY in Formulation P compared to Formulation A or B (peptideYY control formulation; 60 μg peptide YY₃₋₃₆ in 100 ml of phosphatebuffered saline (PBS) at pH 7.4 or 5.0; Bachem AG, King of Prussia,Pa.). On average the cumulative increase in permeation at 120 minutesusing Formulation P exemplary intranasal formulation is about 1195 foldgreater than Formulations A or B controls.

TABLE 3 Influence of Pharmaceutical Formulations Comprising Peptide YYand Intranasal Delivery-Enhancing Agents on Permeation of Peptide YYthrough EpiAirway Cell Membrane by ELISA Assay. Formulation FoldIncrease Peptide YY₃₋₃₆ (60 % Permeation at Time Points (min) Total % inμg/100 ml) 0 15 30 60 120 Permeation Permeability A PBS pH 7.4(Control 1) 0 0.00171 0.00096 0.00451 0.00327 0.01 1 B PBS pH 5.0(Control 2) 0 0.00093 0.00048 0.00042 0.00367 0.01 1 C L-Arginine (10%w/v) 0 0.00119 0.00277 0.00685 0.00566 0.02 2 D Poly-L-Arginine (0.5% 00.00324 0.01587 0.10395 0.49656 0.62 62 w/v) E Gamma-Cyclodextrin 00.00017 0.00042 0.00028 0.0035 0 1 (1% w/v) F α-Cyclodextrin (5% 00.00031 0.000745 0.00147 0.0031 0.01 1 w/v) G Methyl-β-Cyclodextrin 00.00028 0.00038 0.00059 0.01028 0.01 1 (3% w/v) H n-Capric Acid Sodium 00.0004 0.00131 0.00448 0.00821 0.01 1 (0.075% w/v) I Chitosan (0.5% w/v)0 0.00086 0.01098 0.09749 0.82126 0.93 93 J L-α-phosphatidilcholine 00.00934 0.02 0.08507 1.9642 2.08 208 didecanyl (3.5% w/v) KS-Nitroso-N-Acetyl- 0 0.00074 0.0032 0.0688 0.90432 0.98 98Penicillamine (0.5% w/v) L Palmotoyl-DL- 0 0.00378 0.03422 0.151411.31011 1.5 150 Carnitine (0.02% w/v) M Pluronic-127 (0.3% 0 0.000250.00027 0.00066 0.00395 0.01 1 w/v) N Sodium Nitroprusside 0 0.001710.00114 0.00079 0.05492 0.05 5 (0.3% w/v) O Sodium Glycocholate 00.00325 0.00313 0.09023 0.70214 0.8 80 (1% w/v) P F1 Gelatin, DDPC, 00.05864 1.3972 2.9799 7.519 11.95 1195 MBCD, EDTAMTT Assay:

The MTT assays were performed using MTT-100, MatTek kits. 300 mL of theMTT solution was added into each well. Tissue inserts were gently rinsedwith clean PBS and placed in the MTT solution. The samples wereincubated at 37° C. for 3 hours. After incubation the cell cultureinserts were then immersed with 2.0 mL of the extractant solution perwell to completely cover each insert. The extraction plate was coveredand sealed to reduce evaporation. Extraction proceeds overnight at RT inthe dark. After the extraction period was complete, the extractantsolution was mixed and pipetted into a 96-well microtiter plate.Triplicates of each sample were loaded, as well as extractant blanks.The optical density of the samples was then measured at 550 nm on aplate reader (Molecular Devices).

The MTT assay on an exemplary formulation for enhanced nasal mucosaldelivery of peptide YY following the teachings of the instantspecification (e.g., Formulation P) compared to control formulation(Formulations A or B) are shown in Table 4. The results for formulationscomprising peptide YY and one or more intransal delivery enhancingagents, for example, Formulation P (experiment performed in threereplicates) indicate that there is minimal toxic effect of thisexemplary embodiment on viability of the mucosal epithelial tissue.

TABLE 4 Influence of Pharmaceutical Formulations Comprising Peptide YYand Intranasal Delivery-Enhancing Agents on the Viability of EpiAirwayCell Membrane as shown by % MTT Formulations Treatment % MTT A PBS pH .4(Control 1) 100 B PBS pH 5.0 (Control 2) 100 C L-Arginine (10% w/v)91.54 D Poly-L-Arginine (0.5% w/v) 79.39 E Gamma-Cyclodextrin (1% w/v)100 F α-Cyclodextrin (5% w/v) 96.63 G Methyl-β-Cyclodextrin (3% w/v) 100H n-Capric Acid Sodium (0.075% w/v) 100 I Chitosan (0.5% w/v) 100 JL-α-phosphatidilcholine didecanyl (3.5% w/v) 94.25 KS-Nitroso-N-Acetyl-Penicillamine (0.5% w/v) 97.64 LPalmotoyl-DL-Carnitine (0.02% w/v) 91.77 M Pluronic-127 (0.3% w/v) 100 NSodium Nitroprusside (0.3% w/v) 100 O Sodium Glycocholate (1% w/v) 100 PF1: Gelatin, DDPC, MBCD, EDTA 88.75LDH Assay: The LDH assay on an exemplary formulation for enhanced nasalmucosal delivery of peptide YY following the teachings of the instantspecification (e.g., Formulation P) are shown in Table 5. The resultsfor three replicates of Formulation P indicate that there is minimaltoxic effect of this exemplary embodiment on viability of the mucosalepithelial tissue.

TABLE 5 Influence of Pharmaceutical Formulations Comprising Peptide YYand Intranasal Delivery-Enhancing Agents on the Viability of EpiAirwayCell Membrane as shown by % Dead Cells (LDH Assay) % Dead FormulationsTreatment Cells A PBS pH .4 (Control 1) 1.0 B PBS pH 5.0 (Control 2) 1.1C L-Arginine (10% w/v) 0.8 D Poly-L-Arginine (0.5% w/v) 1.4 EGamma-Cyclodextrin (1% w/v) 0.8 F α-Cyclodextrin (5% w/v) 0.7 GMethyl-β-Cyclodextrin (3% w/v) 0.8 H n-Capric Acid Sodium (0.075% w/v)1.3 I Chitosan (0.5% w/v) 0.7 J L-α-phosphatidilcholine didecanyl (3.5%w/v) 1.2 K S-Nitroso-N-Acetyl-Penicillamine (0.5% w/v) 0.7 LPalmotoyl-DL-Carnitine (0.02% w/v) 0.8 M Pluronic-127 (0.3% w/v) 1.0 NSodium Nitroprusside (0.3% w/v) 0.6 O Sodium Glycocholate (1% w/v) 0.8 PF1: Gelatin, DDPC, MBCD, EDTA 2.0

EXAMPLE 3 Formulation P (Peptide YY) of the Present Invention inCombination with Triamcinolone Acetonide Corticosteroid Improves CellViability

The present example provides an in vitro study to determine thepermeability and reduction in epithelial mucosal inflammation of anintranasally administered peptide YY, for example, human peptide YY, incombination with a steroid composition, for example, triamcinoloneacetonide, and further in combination with one or more intranasaldelivery-enhancing agents. The study involves determination ofepithelial cell permeability by TER assay and reduction in epithelialmucosal inflammation as measured by cell viability in an MTT assay byapplication of an embodiment comprising peptide YY and triamcinoloneacetonide.

Formulation P (see Table 1 above) is combined in a formulation withtriamcinolone acetonide at a dosage of 0.5, 2.0, 5.0, or 50 μg. Normaldose of triamcinolone acetonide, (Nasacort®, Aventis Pharmaceuticals)for seasonal allergic rhinitis, is 55 μg per spray. Formulation P incombination with triamcinolone acetonide corticosteroid improves cellviability as measured by the MTT assay, while maintaining epithelialcell permeability as measured by TER and ELISA assays.

According to the methods and formulations of the invention, measurementof permeability of Formulation P in the presence or absence oftriamcinolone acetonide is performed by transepithelial electricalresistance (TER) assays in an EpiAirway™ cell membrane. TER assays ofFormulation P plus triamcinolone acetonide at a concentration of 0.5,2.0, 5.0, or 50 μg per spray indicate that peptide YY permeability didnot decrease and was equal to permeability of Formulation P alone.Formulation P plus triamcinolone acetonide at a triamcinolone acetonideconcentration between 0 and 50 μg per spray is typically, at least10-fold to 100-fold greater than permeability of Formulations A or B(peptide YY control).

According to the methods and formulations of the invention, measurementof permeability of Formulation P in the presence or absence oftriamcinolone acetonide is performed by ELISA assay in an EpiAirway™cell membrane. Similar to the TER assay above, ELISA assay ofFormulation P plus triamcinolone acetonide at a concentration of 0.5,2.0, 5.0, or 50 μg per spray indicate that peptide YY permeability didnot decrease and was equal to permeability of Formulation P alone.Formulation P plus triamcinolone acetonide at a triamcinolone acetonideconcentration between 0 and 50 μg per spray is typically greater thanpermeability of Formulations A or B (peptide YY control).

According to the methods and formulations of the invention, MTT assaymeasured cell viability of Formulation P in the presence or absence oftriamcinolone acetonide. Typically, addition of triamcinolone acetonide(at a concentration of 0.5, 2.0, 5.0, or 50 μg per spray) to FormulationP improves cell viability compared to Formulation P in the absence oftriamcinolone acetonide.

Addition of triamcinolone acetonide to Formulation P increases cellviability and maintains epithelial permeability as measured by TER assaycomparable to Formulation P in the absence of triamcinolone acetonide.

Reduction in epithelial mucosal inflammation of an intranasallyadministered peptide YY is accomplished with an intranasal formulationof peptide YY in combination with one or more steroid or corticosteroidcompound(s) typically high potency compounds or formulations, but alsoin certain cases medium potency, or low potency compounds orformulations. Overall potency (equivalent dosages) of high, medium, andlow potency steroids are given. Typically, an intranasal formulation ofpeptide YY in combination with a high potency steroid compositionincludes, but is not limited to, betamethasone (0.6 to 0.75 mg dosage),or dexamethasone (0.75 mg dosage). In an alternative formulation, anintranasal formulation of peptide YY in combination with a mediumpotency steroid composition includes, but is not limited to,methylprednisolone (4 mg dosage), triamcinolone (4 mg dosage), orprednisolone (5 mg dosage). In a further alternative formulation, anintranasal formulation of peptide YY in combination with a low potencysteroid composition includes, but is not limited to hydrocortisone (20mg dosage) or cortisone (25 mg dosage).

EXAMPLE 4 Bioavailability and Bioactivity of Three Different Doses ofPeptide YY (PYY) Via Nasal Mucosal Administration to Healthy Subjects toMeasure Appetite Control

Study Synopsis

The present example provides a non-blinded study to determine the uptakeof intranasally administered peptide YY into the blood serum in healthymale volunteers. The study involves administration of peptide YY nasalformulation, as described above to evaluate the absorption and toleranceof the peptide YY nasal formulation

Twelve healthy male subjects, age 18-50, are enrolled in the study. Eachreceives one intranasal dose of the test formulation. Each subjectvisits the clinical site three times in a 3-week period. These visitsconsist of a screening visit, one dosing visit, and a final visit.Demographic data, subject initials, gender, age, race and statement ofnon-smoking status is recorded at the time of screening. A completemedical history and physical examination including electrocardiogram,vital signs, height and weight, and clinical laboratory evaluations isconducted at screening and when the subject completes the study.

The proposed study involves administration of one reformulated productof intranasal formulation of peptide YY as follows:

-   -   Control Product/Formulation B: Nasal spray=40 pmol PYY₃₋₃₆/0-1        ml spray (one 0.1 ml spray in each nostril each day; in PBS, pH        5.0). Formulation PYY₃₋₃₆ (Bachem AG, King of Prussia, Pa.) One        0.1 ml spray to one nostril every day, alternating from left        nostril to right    -   Test Formulation P Product: Nasal spray=2.6 mg/0.1 ml spray.        (Formulation P: PYY₃₋₃₆, 60 μg per 100 ml; Gelatin, DDPC, MBCD,        EDTA as described in Table 1). One 0.1 ml spray in each nostril        each day; or one 0.1 ml spray to one nostril every day,        alternating from left nostril to right.

The absorption and tolerance results of all test products tested will betabulated and analyzed for C_(max), t_(max) and AUC. Data resulting formthe study will be compared to the pharmacokinetic parameters in theavailable literature and to the data from the peptide YY studies usingFormulation B control and Formulation P.

For each preparation, 7 mL blood samples will be drawn at 0 (prior todose), 10, 20, 30, 45, 60, 75, 90, 120, 180 and 240 minutes post dosinginto appropriate vacutainers.

Serum anti-human peptide YY antibodies will be measured at the screeningand final visits.

On the day of dosing, subjects' vital signs (blood pressure, pulse,respiration rate and body temperature) will be monitored before dosingand post dosing at 15, 30, 45, 60, 75, 90, 120 and 240 minutes postdosing and prior to discharge.

The nasal examination will be performed by qualified personnel atpre-dosing, 15, 30, 45, 60, 75, 90, 120 and 240 minutes and prior todischarge from the visit.

The results of the study will be evaluated for each test dose for safetyand absorption. If administration of the dose results in a grading scaleof 3 (based on the Common Toxicity Criteria [CTC]) for any of theparameters observed, the study arm will be discontinued.

The intent of the study, the study protocol, and the Informed ConsentForm to be used in the study is approved in writing by the IRB prior toinitiation of the study.

Subject Inclusion Criteria

The following inclusion criteria are used:

-   -   Healthy male subjects.    -   Age 18-50.    -   Non-smokers (greater than 6 months).    -   For whom administration of peptide YY is not contraindicated        (such as known hypersensitivity to the product or any of the        constituents).

The male subjects have a normal nasal mucosa. Demographic data, subjectinitials, gender, age, race and statement of non-smoking status arerecorded at screening. A complete medical history and physicalexamination including electrocardiogram, vital signs, height and weight,and the following laboratory tests are conducted at screening and whenthe subject completes the study: Blood Chemistry, Thyroid FunctionTests, Hematology, Urinalysis, Drug Screens.

Subject Exclusion Criteria

The following exclusion criteria are used:

-   -   Subjects with a history of hypersensitivity to natural or        recombinant peptide YY or any other component of the Formulation        P (Gelatin, DDPC, MBCD, EDTA as described in Table 1).    -   Subjects with active neoplasia.    -   Subjects with glucose intolerance, diabetes mellitus or a family        history of diabetes.    -   Subjects with thyroid hormone abnormalities.    -   Subjects currently taking glucocorticoids.    -   Subjects with clinically significant nasal abnormalities.    -   Subjects with history of nosebleeds or allergic rhinitis.    -   Subject with history of alcoholism or drug abuse.    -   Subject with psychiatric disorders.    -   Subjects with acute critical illness due to complications        following open heart or abdominal surgery, multiple accidental        trauma or patients having acute respiratory failure.        Dosing

Before dosing, all subjects will be given an orientation of the properdosing technique and general conduct of the study.

-   -   Physical Activity: Avoid vigorous exertion for 3 hours after        dose.    -   Confinement: Subjects will be confined immediately prior to the        first draw and at least until the last blood draw is completed.        Subjects may be confined longer at the discretion of the        Principal Investigator.    -   Fasting: Volunteers are not required to fast before the study.        However, during the study they may not eat until after the        90-minute blood draw time point.    -   Meals: Meals may be provided after the 90-minute blood sample.    -   Fluid Intake: Hot and cold carbonated liquids are prohibited for        90 minutes before and 90 minutes after dosing (water allowed).    -   Environmental Conditions: Subjects will be in a smoke-free        environment at time of dosing and/or during study confinement.        Full resuscitative facilities will be immediately available.    -   Concurrent Medication: Subjects will be instructed to take no        antibiotics for at least 2 days and no medications including        alcohol, monoamine oxidase (MAO) inhibitors, sedatives,        antihistamines, psychotropic drugs and any OTC products for at        least three days prior to the start of the study. They will also        be informed to take no intranasal medications (including        intranasal OTC) for three days prior to or during the study        except those administered as per the study protocol.

The intranasal formulation is manufactured by Nastech PharmaceuticalClinical Supply department under GMP compliance. The intranasalformulation is either Formulation B (control) or Formulation P, asdescribed above. The dosage comprises one 0.1 ml spray in each nostrileach day; or one 0.1 ml spray to one nostril every day, alternating fromleft nostril to right.

When receiving the nasal spray, the subject is seated and instructed togently blow his nose before dosing. During dosing, the other nostrilmust be closed with the forefinger. Subjects are instructed to tilttheir heads slightly back for dosing and to return their heads to anupright position while sniffing in gently immediately following dosing.Subjects must avoid additional sniffing and must remain in a seatedposition with head upright for minutes after dosing. Subjects mustinform the staff if they sneeze or if the product drips out of theirnose.

The blood samples are collected in 7 mL vacutainers and centrifuged atroom temperature for not less than 8 minutes at 1,500 rpm after at least30 minutes have elapsed from the time of blood draw. At least 1.2 mL ofserum is pipetted into the first of two prelabeled polypropylene tubes,with the remainder pipetted into the second tube. Both tubes are frozenpromptly and stored at −10° C. for no more than 30 days until analysis.

The second sample is retained by the Investigator until the studymonitor notifies him/her of the appropriate disposition.

All subjects are monitored throughout the confinement portion of thestudy. Blood pressure, respiration rate, pulse, and body temperature areobtained prior to dosing and as scheduled following dosing. Dosingproceeds as authorized by the medical investigator who will be availableon-site and/or by pager throughout the study.

Serum drug concentrations are measured using a validated ELISA method.The concentration at each sampling time and the appropriatepharmacokinetic parameters are reported.

On the day of dosing, subjects' vital signs (blood pressure, pulse,respiration rate and body temperature are monitored before dosing andpost dosing at 15, 30, 45, 60, 75, 90, 120 and 240 minutes post dosingand prior to discharge.

Nasal Mucosal Examinations

The investigator, or a medically qualified designee(Sub-Investigator/Nurse Practitioner), visually examines the nasalmucosa of all subjects. On the day of dosing these examinations areperformed immediately before the intranasal dosing and at 15, 30, 45,60, 75, 90, 120, and 240 minutes after dosing and prior to dischargefrom the visit.

Observations are made upon examination of the nasal mucosa which coversthe septum and turbinates. The investigator notes upon examination thecolor (redness) and swelling, bleeding or exudates. If exudates arepresent, they are noted for character, clear, mucopusulent or pusulent.The nasal septum is examined for any deviation, inflammation orperforation of the septum. The septum is observed for epistaxis. Anyabnormalities such as ulcers or polyps is also be documented.

All observations are recorded in the adverse event forms in the CaseReport Forms. Each subject completes a nasal tolerance questionnaire onthe formulations administered.

Absorption Data Evaluation

All absorption data will be plotted for individual subjects as well asfor the averaged data. The C_(max), t_(max) and the bioavailability(measured as area under the individual serum peptide YY time curves,AUC) of the test products are evaluated with the goal of comparing theaforementioned pharmacokinetic parameters for intransal formulations,Formulation B control or Formulation P, as described above.

Statistics: Determination of AUC

The areas under the individual serum GH concentration vs. time curves(AUC) were calculated according to the linear trapezoidal rule and withaddition of the residual areas. A decrease of 23% or an increase of 30%between two dosages would be detected with a probability of 90% (type IIerror β=10%). The rate of absorption was estimated by comparison of thetime (t_(max)) to reach the maximum concentration (C_(max)). BothC_(max) and t_(max) were analyzed using non-parametric methods.Comparisons of the pharmacokinetics of subcutaneous, intravenous, andintranasal peptide YY administration were performed by analysis ofvariance (ANOVA). For pairwise comparisons a Bonferroni-Holmessequential procedure was used to evaluate significance. Thedose-response relationship between the three nasal doses was estimatedby regression analysis. P<0.05 was considered significant. Results aregiven as mean values +/−SEM. Laursen, et al., Eur. J. Endocrinology 135:309-315, 1996, incorporated herein by reference.

Results:

Due to its unique characteristics, the intranasal administration ofpharmaceutical formulations of the present invention comprising peptideYY and one or more intranasal delivery-enhancing agents offers manyadvantages in terms of providing absorption of macromolecular drugswhich are either not absorbed or variably absorbed after oraladministration or absorbed more slowly following intramuscular orsubcutaneous injection. No non-injectable products of peptide YY arecurrently available. Pulmonary administration has achieved some successbut has disadvantages including patient inconvenience and questionablepulmonary safety.

According to the methods and formulations of the invention,pharmacokinetic data for intranasal delivery of peptide YY in apharmaceutical formulation of the present invention (e.g., FormulationP) is compared to subcutaneous delivery of a control formulation ofpeptide YY (Formulation B).

The results exemplify bioavailability of peptide YY achieved by themethods and formulations herein, e.g., as measured by area under theconcentration curve (AUC) in blood serum, CNS, CSF or in anotherselected physiological compartment or target tissue. According to themethods and formulations of the invention, bioavailability of peptide YYwill be, typically, AUC_(0-8 hr) for peptide YY of approximately 100pmol·hr/L of blood plasma or CSF, AUC_(0-8 hr) for peptide YY ofapproximately 200 pmol·hr/L of blood plasma or CSF, or AUC_(0-8 hr) forpeptide YY up to approximately 400 pmol·hr/L of blood plasma or CSF.

According to the methods and formulations of the invention, relativebioavailability as measured by area under the concentration curve (AUC)for an exemplary intranasal formulation (Formulation P) of peptide YY ofthe present invention is typically 5% to 6% relative to subcutaneousadministration under comparable experimental conditions. This result iscompared to relative bioavailability of a control formulation (humanpeptide YY; Formulation B) is typically 1% to 3% for intranasal deliveryof a prior art formulation of peptide YY relative to subcutaneousadministration under comparable experimental conditions. According tothe methods and formulations of the invention, the exemplary formulationadministered intranasally provides time to maximal plasma concentrationof peptide YY typically between 0.3 to 1.0 hours. These results arefully consistent with the foregoing disclosure.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications are comprehended bythe disclosure and may be practiced without undue experimentation withinthe scope of the appended claims, which are presented by way ofillustration not limitation.

What is claimed is:
 1. A composition comprising a Y2-receptor selectiveagonist, a solubilizing agent, a chelating agent, andL-α-phosphatidylcholine didecanoyl (DDPC).
 2. The composition of claim1, wherein the Y2-receptor selective agonist is PEGylated.
 3. Thecomposition of claim 1, wherein the Y2-receptor selective agonist isselected from the group consisting of neuropeptide Y (SEQ ID NO:795),([¹²⁵I]-Tyr)-neuropeptide Y (SEQ ID NO:802), biotinyl-neuropeptide Y(SEQ ID NO:803), (Leu³¹,Pro³⁴)-neuropeptide Y (SEQ ID NO:804),(D-Trp³²)-neuropeptide Y (SEQ ID NO:805), (Tyr(Me)²¹)-neuropeptide Y(SEQ ID NO:806), (A1a³¹,Aib³²)-neuropeptide Y (SEQ ID NO:807),(Leu³¹,Pro³⁴)-neuropeptide Y (SEQ ID NO:808), (Pro³⁴)-neuropeptide Y(SEQ ID NO:809), (D-Trp³²)-neuropeptide Y (SEQ ID NO:810), neuropeptideY₁₋₂₄ amide (SEQ ID NO:811), (Cys²)-neuropeptideY₁₋₄-8-aminooctanoyl-(D-Cys²⁷)-neuropeptide Y₂₅₋₃₂ (SEQ ID NO:812),neuropeptide Y₂₋₃₆ (SEQ ID NO:813), neuropeptide Y₃₋₃₆ (SEQ ID NO:814),neuropeptide Y₁₃₋₃₆ (SEQ ID NO:815), (Leu³¹,Pro³⁴)-neuropeptide Y₁₃₋₃₆(SEQ ID NO:816), neuropeptide Y₁₈₋₃₆ (SEQ ID NO:817), pancreaticpolypeptide₁₋₁₇-A1a³¹,Aib³²)-neuropeptide Y₁₈₋₃₆ (SEQ ID NO:818),neuropeptide Y₂₂₋₃₆ (SEQ ID NO:819),Tyr-Lys-Gly-Arg-(Glu²⁶,Lys²⁹,Pro³⁴)-neuropeptide Y₂₆₋₃₆ (SEQ ID NO:820),(D-Tyr^(27,36),D-Thr³²)-neuropeptide Y₂₇₋₃₆ (SEQ ID NO:821),((Cys³¹,Nva³⁴)-neuropeptide Y₂₇₋₃₆)₂(SEQ ID NO:822),(Pro³⁰,Tyr³²,Leu³⁴)-neuropeptide Y₂₈₋₃₆ (SEQ ID NO:823),(His³²,Leu³⁴)-neuropeptide Y₃₂₋₃₆ (SEQ ID NO:824),([¹²⁵I]-Tyr)-(Leu³¹,Pro³⁴)-neuropeptide Y (SEQ ID NO:825),(Gly¹,Ser^(3,22),Gln^(4,34),Thr⁶,Ala¹⁹,Tyr²¹,Ala^(23,31),Aib³²)-pancreatic polypeptide (human) (SEQ ID NO:826),and Y2A (N-acetyl [Leu²⁸, Leu³¹]NPY₂₄₋₃₆) (SEQ ID NO:827).
 4. Thecomposition of claim 1, wherein the solubilizing agent is selected fromthe group consisting of hydroxypropyl-β-cyclodextrin,sulfobutylether-β-cyclodextrin, and methyl-β-cyclodextrin.
 5. Thecomposition of claim 1, wherein the chelating agent is selected from thegroup consisting of ethylene diamine tetraacetic acid (EDTA) andethylene glycol tetraacetic acid (EGTA).
 6. The composition of claim 1,wherein the composition is a dry powder.
 7. The composition of claim 1,further comprising a pH control agent.
 8. The composition of claim 7,wherein the pH control agent is selected from the group consisting ofarginine, sodium hydroxide, glycine, hydrochloric acid, and citric acid.9. The composition of claim 1, wherein the composition is an aqueouscomposition having a pH from about 3 to about
 6. 10. The composition ofclaim 1, wherein the composition is an aqueous composition having a pHof about
 5. 11. The composition of claim 1, wherein the composition isan aqueous composition having a pH of about
 4. 12. The composition ofclaim 1, further comprising a preservative.
 13. The composition of claim12, wherein the preservative is selected from the group consisting ofmethyl-p-hydroxybenzoate, propyl-p-hydroxybenzoate, phenol, methylparaben, paraben, m-cresol, thiomersal, benzylalkonimum chloride, andchlorobutanol.
 14. The composition of claim 1, further comprising arelease-enhancing agent.
 15. The composition of claim 14, wherein therelease-enhancing agent is polyethylene glycol (PEG).